We describe a female patient with developmental delay, dysmorphic features and multiple congenital anomalies who presented a normal G-banded karyotype at the 550-band resolution. Array and multiplex-ligation probe amplification (MLPA) techniques identified an unexpected large unbalanced genomic aberration: a 17.6Mb deletion of 9p associated to a 14.8 Mb duplication of 20p. The deleted 9p genes, especially CER1 and FREM1, seem to be more relevant to the phenotype than the duplicated 20p genes. This study also shows the relevance of using molecular techniques to make an accurate diagnosis in patients with dysmorphic features and multiple anomalies suggestive of chromosome aberration, even if on G-banding their karyotype appears to be normal. Fluorescence in situ hybridization (FISH) was necessary to identify a masked balanced translocation in the patient's mother, indicating the importance of associating cytogenetic and molecular techniques in clinical genetics, given the implications for patient management and genetic counseling.

OBJECTIVE

To investigate the involvement of transcription factor Foxa2 in cardiac differentiation in P19 embryonal carcinoma cells and its molecular mechanism.

METHODS

P19 cells were induced to differentiate into cardiomyocytes by adding dimethyl sulfoxide (DMSO) into the culture medium of their embryoid bodies (EBs). The mRNA levels of pluripotency markers of embryonic pluripotent stem cells, cardiac differentiation related genes, and Foxa2 in the cell samples at different time points of cardiac differentiation were detected by reverse transcription PCR (RT-PCR). Differentiated and mature cardiomyocytes were identified by immunofluorescence. Eukaryotic expression plasmid pCMV-rFoxa2 (rat Foxa2) was transfected into P19 cells, and clonal populations of P19 cells that stably expressed green fluorescence protein (GFP)-rFoxa2 were isolated to enhance the expression levels of Foxa2 in P19 cells. The mRNA and protein levels of pluripotency markers and cardiac differentiation related genes in the above cell samples were detected by RT-PCR and Western blot. The mRNA levels of cardiac differentiation related genes in EBs differentiation system were also examined.

RESULTS

P19 cells differentiated into cardiomyocytes in the presence of DMSO, accompanied by stimulated expression of Foxa2. Transfection of pCMV-rFoxa2 plasmids into P19 cells upregulated rFoxa2 expression transiently and activated the transcription of its downstream cardiac inducer Cerberus1 (Cer1). The expression of pluripotency marker Nanog was suppressed and the expression of cardiac inducer Sonic Hedgehog (Shh) was elevated in GFP-rFoxa2 P19 cells. The expression of Cer1 and cardiac muscle marker actin, alpha cardiac muscle 1 (Actc1) was upregulated in EBs of GFP-rFoxa2 P19 cells.

CONCLUSIONS

Foxa2 participates in cardiac differentiation in P19 embryonal carcinoma cells. Foxa2 may inhibit Nanog expression and stimulate the expression of Cer1 and Shh directly during cardiac differentiation in P19 cells in the presence of DMSO.

BACKGROUND

Leaf epicuticular wax is an important functional trait for physiological regulation and pathogen defense. This study tests how selective pressure may have forced the trait of leaf abaxial epicuticular wax crystals (LAEWC) and whether the presence/absence of LAEWC is associated with other ecophysiological traits. Scanning Electron Microscopy was conducted to check for LAEWC in different Lithocarpus species. Four wax biosynthesis related genes, including two wax backbone genes ECERIFERUM 1 (CER1) and CER3, one regulatory gene CER7 and one transport gene CER5, were cloned and sequenced. Ecophysiological measurements of secondary metabolites, photosynthesis, water usage efficiency, and nutrition indices were also determined. Evolutionary hypotheses of leaf wax character transition associated with the evolution of those ecophysiological traits as well as species evolution were tested by maximum likelihood.

RESULTS

Eight of 14 studied Lithocarpus species have obvious LAEWC appearing with various types of trichomes. Measurements of ecophysiological traits show no direct correlations with the presence/absence of LAEWC. However, the content of phenolic acids is significantly associated with the gene evolution of the wax biosynthetic backbone gene CER1, which was detected to be positively selected when LAEWC was gained during the late-Miocene-to-Pliocene period.

CONCLUSIONS

Changes of landmass and vegetation type accelerated the diversification of tropical and subtropical forest trees and certain herbivores during the late Miocene. As phenolic acids were long thought to be associated with defense against herbivories, co-occurrence of LAEWC and phenolic acids may suggest that LAEWC might be an adaptive defensive mechanism in Lithocarpus.

The Arabidopsis ECERIFERUM1 (CER1) protein is a decarbonylase that converts fatty acid metabolites into alkanes. Alkanes are components of waxes in the plant cuticle, a waterproof barrier serving to protect land plants from both biotic and abiotic stimuli. CER1 enzymes can be used to produce alternative and sustainable hydrocarbons in eukaryotic systems. In this report we identified 193 CER1 and 128 CER3 sequences from 56 land plants respectively. CER1 and CER3 proteins have high amino acid similarity and both are involved in alkane synthesis in Arabidopsis. The common homologues of CER1 and CER3 genes were identified in three species of chlorophytes, which may be one of the earliest plant taxa that possess CER1 and CER3 genes. To facilitate potential applications, the 3-dimensional structure and conserved motifs of CER1 proteins were also characterized. CER1 and CER3 proteins are structurally similar, but CER1 proteins have more conserved histidine-containing motifs common to fatty acid hydroxylases and stearoyl-CoA desaturases. There was no significant loss or gain of protein motifs after ancient and recent duplications, suggesting that varied properties of CER1 proteins may be associated with less-conserved regions. Among 56 land plants, the codon-based assessments of selection modes revealed that neither entire proteins nor individual amino acids of CER1 proteins were significantly subjected to positive selection, indicating that CER1 proteins are highly conserved throughout evolution.

ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3) are key genes in synthesis of alkanes, a major component of cuticular waxes in land plants. The genes share extensive similarity, including the N-terminal (ERG3/FAH) and C-terminal (WAX2) domains. This study traces the origin, evolutionary history, phylogenetic relationships and variation in copy number of the two genes within and beyond the Viridiplantae (green plants). Protein homologs of both CER1 and CER3 were identified across most Embryophyta (land plants), a single homolog (CER1/3) in charophytes and prasinophytes, and none in other green, red or brown algae. Ancestral state reconstructions in 100 sequenced Archaeplastida using presence/absence of CER1/3 family genes revealed that the CER1/3 gene probably originated in the common ancestor of Viridiplantae. Phylogenetic analysis of CER1 and CER3 protein sequences from 146 plant species strongly suggests that the two genes originated by duplication of CER1/3 in the ancestral embryophyte. The evolution of CER1 and CER3 genes involved differential divergence of the two domains. Outside Embryophyta, CER1/3 similar sequences identified in diatoms and a cryptophyte, were the closest relatives of the CER1/3 family proteins. Proteins harbouring WAX2-wxAR (WAX2 associated region) similar regions were identified in proteins of bacteria, Archaea, cryptophytes, dinoflagellates and Stramenopiles. The independent existence of both ERG3/FAH and WAX2-wxAR domains in diverse lineages strongly points to the origin of CER1/3 gene in green plants by the fusion of pre-existing domains.

Keywords: ECERIFERUM1 (CER1); ECERIFERUM3 (CER3); WAX2 domain; alkanes; cuticular waxes.

BACKGROUND

Epiblasts occur at the last pluripotent stage of embryonic development and are important in elucidating how the three germ layers are formed. However, little is known of the molecular mechanisms of their development. We have shown that LIM homeobox 1 (Lhx1) was involved in epiblast development in embryonic stem cells, especially meso- and endodermal differentiation. However, since epiblasts in embryoid bodies spontaneously develop into a further stage, it is difficult to study their development in this system.

METHODS

Mouse embryonal carcinoma P19 cells which have properties similar to those of epiblasts provided new avenues of investigation into the regulatory mechanism of epiblasts.

RESULTS

Overexpression of Lhx1 in P19 cells induced expression of organizer marker genes (Cer1, Gsc) and endoderm marker genes (Gata6, Foxa2, Sox17) but not extra-embryonic endoderm marker genes (Sox7 or Hnf4alpha).

CONCLUSIONS

This study suggested that Lhx1 overexpression caused P19 cells to differentiate into an endodermal lineage. Thus, P19 cells and their derivatives can be a useful model system to study how the three germ layers are formed.

BACKGROUND

Understanding how embryos specify asymmetric axes is a major focus of biology. While much has been done to discover signaling pathways and transcription factors important for axis specification, comparatively little is known about how epigenetic regulators are involved. Epigenetic regulators operate downstream of signaling pathways and transcription factors to promote nuclear processes, most prominently transcription. To discover novel functions for these complexes in axis establishment during early embryonic development, we characterized phenotypes of a mouse knockout (KO) allele of the chromatin remodeling Ino80 ATPase.

RESULTS

Ino80 KO embryos implant, but fail to develop beyond the egg cylinder stage. Ino80 KO embryonic stem cells (ESCs) are viable and maintain alkaline phosphatase activity, which is suggestive of pluripotency, but they fail to fully differentiate as either embryoid bodies or teratomas. Gene expression analysis of Ino80 KO early embryos by in situ hybridization and embryoid bodies by RT-PCR shows elevated Bmp4 expression and reduced expression of distal visceral endoderm (DVE) markers Cer1, Hex, and Lefty1. In culture, Bmp4 maintains stem cell pluripotency and when overexpressed is a known negative regulator of DVE differentiation in the early embryo. Consistent with the early embryo, we observed upregulated Bmp4 expression and down-regulated Cer1, Hex, and Lefty1 expression when Ino80 KO ESCs are differentiated in a monolayer. Molecular studies in these same cells demonstrate that Ino80 bound to the Bmp4 promoter regulates its chromatin structure, which correlates with enhanced SP1 binding. These results in combination suggest that Ino80 directly regulates the chromatin structure of the Bmp4 promoter with consequences to gene expression.

CONCLUSIONS

In contrast to Ino80 KO differentiated cells, our experiments show that undifferentiated Ino80 KO ESCs are viable, but fail to differentiate in culture and in the early embryo. Ino80 KO ESCs and the early embryo up-regulate Bmp4 expression and down-regulate the expression of DVE markers Cer1, Hex and Lefty1. Based on this data, we propose a model where the Ino80 chromatin remodeling complex represses Bmp4 expression in the early embryo, thus promoting DVE differentiation and successful proximal-distal axis establishment. These results are significant because they show that epigenetic regulators have specific roles in establishing embryonic axes. By further characterizing these complexes, we will deepen our understanding of how the mammalian embryo is patterned by epigenetic regulators.

Objective: To evaluate the clinical values of 4 types of ceramides (Cer1, Cer2, Cer3, Cer4) in the coronary artery stenosis, clinical diagnosis and risk prediction. Methods: A total of 890 patients with coronary heart disease (CHD) in Beijing Anzhen Hospital between March 2018 and August 2018 were enrolled. The relationships between different degrees of coronary artery stenosis and ceramide levels was investigated. Diagnostic value of ceramides on acute myocardial infarction was analyzed. Meanwhile, Major adverse cardiac and cerebrovascular events (MACCE) in 1 year after discharging were collected to evaluate the predictive value of ceramides on risk of CHD and stroke. Results: This study showed that there were no significant differences of ceramide levels in CHD patients with different degrees of coronary artery stenosis (P>0.05), and the area under receiver operating characteristic (ROC) curve in the diagnosis of acute myocardial infarction patients was 0.725. Conclusions: Ceramide is proved to be helpful in the diagnosis of acute myocardial infarction and MACCE prediction. The relationships between ceramide and degrees of coronary artery stenosis as well as the prognosis of CHD need further elucidation.

Borage oil [BO: 40.9% linoleic acid (LNA) and 24.0% γ-linolenic acid (GLA)] reverses disrupted epidermal lipid barrier in essential fatty acid deficiency (EFAD). We determined the effects of BO on lamellar body (LB) content and LNA and GLA incorporation into epidermal ceramide 1 (CER1) and epidermal ceramide 2 (CER2), major barrier lipids. EFAD was induced in guinea pigs by a diet of 6% hydrogenated coconut oil (HCO) for 10 weeks (group HCO) or 8 weeks followed by 6% BO for 2 weeks (group HCO + BO). LB content and LNA and GLA incorporation into CER1 were higher in group HCO + BO than in group HCO. Small but significant levels of LNA, GLA, and their C20-metabolized fatty acids [dihomo-γ-linolenic acid (DGLA) and arachidonic acid (ARA)] were incorporated into CER2, where ARA was detected at a level lower than LNA, but DGLA incorporation exceeded that for GLA in group HCO + BO. Dietary BO enhanced LB content and differential incorporation of GLA into CER1 and DGLA into CER2.

Keywords: Borage oil; Dihomo-γ-linolenic acid amide-linked ceramide 2; Epidermis; Lamellar body; γ-Linolenic acid ester-linked ceramide 1.

The short arm of chromosome 3 is frequently altered in human cancers of different tissue origin. Certain regions on the chromosome arm 3p have been defined by deletion studies in human cancer cells and tissues. Also, regions at 3p are eliminated in microcell hybrids parallel to increased tumorigenicity in immunosuppressed mice. We have analysed chromosome instability and several genes involved in tumor pathogenesis at 3p, such as VHL, RIITGFB, CATNB, MLH1 and FHIT. By studying eleven tumor types, we have shown that the importance of CER1 (common eliminated region 1) transgresses tissue specificity. Comparative studies on losses of VHL, FHIT/FRA3B and CER1 show that the CER1 region is preferably lost in human tumors. Alterations of FHIT are associated with reduced survival of breast and colon cancer patients. The FHIT gene is located at the constitutive fragile region, FRA3B. Chromosome 3, particularly FHIT, is unstable in breast cancer patients who have germ line mutation in the BRCA2 gene. The chromosome instability in BRCA2 tumors reflects the DNA repair mechanism of the gene product Brca2. It can be concluded that our results reflect a synergism of tumor suppressor gene (TSG) losses at the chromosome 3p region in relation to the biological behavior of tumor cells and tumor pathogenesis.

Oleocellosis is a physiological disorder causing blemishes on fruit surface. This study investigated the influence of oleocellosis on the membrane fatty acids and wax in lemon fruit rinds at the morphological, physiological, metabolic and molecular levels by using a variety with a high incidence rate of oleocellosis (green lemon). Oleocellosis-damaged rinds showed loose and flaky wax layers with more fissures on the surface, as well as higher contents of C16 and C18 fatty acids and very long chain (VLC) fatty alkanes while lower contents of VLC fatty aldehydes. The main differentially expressed genes, including FabZ, FAD2 and SAD6 involved in the accumulation of C16 and C18 fatty acids and CER1 involved in the transformation of VLC fatty aldehydes to VLC fatty alkanes, were up-regulated by oleocellosis. These results indicate that oleocellosis accelerates the accumulation of membrane free fatty acids and transformation of VLC fatty aldehydes to VLC fatty alkanes.

Keywords: Constituents; Epicuticular wax; Fatty acid; Gene expression; Green lemon; Oleocellosis.

Constitutional ring chromosome 9, r(9), is a rare chromosomal disorder. Cytogenomic analyses by karyotyping, array comparative genomic hybridization (aCGH) and whole genome sequencing (WGS) were performed in a patient of r(9). Karyotyping detected a mosaic pattern of r(9) and monosomy 9 in 83% and 17% of cells, respectively. aCGH detected subtelomeric deletions of 407 kb at 9p24.3 and 884 kb at 9q34.3 and an interstitial duplication of 5.879 Mb at 9q33.2q34.11. WGS revealed double strand breaks (DSBs) at ends of 9p24.3 and 9q34.3, inverted repeats at ends of subtelomeric and 9q33.2q34.11 regions, and microhomology sequences at the junctions of this r(9). This is the first report of r(9) analyzed by WGS to delineate the mechanism of ring chromosome formation from repairing of subtelomeric DSBs. The loss of telomeres by subtelomeric DSBs triggered inverted repeats induced intra-strand foldback and then microhomology mediated synthesis and ligation, which resulted in the formation of this r(9) with distal deletions and an interstitial duplication. Review of literature found seven patients of r(9) with clinical and cytogenomic findings. These patients and the present patient were registered into the Human Ring Chromosome Registry and a map correlating critical regions and candidate genes with relevant phenotypes was constructed. Variable phenotypes of r(9) patients could be explained by critical regions and genes of DOCK8, DMRT, SMARCA2, CD274, IL33, PTPRD, CER1, FREM1 for 9p deletions, and the EHMT1 gene for 9q34 deletion syndrome. This interactive registry of r(9) could provide information for cytogenomic diagnosis, genetics counseling and clinical management.

Keywords: clinical and cytogenomic findings; double stand breaks repairing; mechanism of ring chromosome formation; ring chromosome 9; whole genome sequencing.

The generation of germ cells from embryonic stem cells in vitro has current historical significance. Western blot, qPCR, immunofluorescence and flow cytometry assays were used to investigate the differences in expression levels of totipotency and specific markers for Wnt regulation and the related signalling pathways during primordial germ cell-like cell (PGCLC) induction and differentiation. During PGCLC induction, activation of WNT3a increased the expression of NANOG, SOX2 and OCT4, but Mvh, DAZL, Blimp1, TFAP2C, Gata4, SOX17, EOMES, Brachyury and PRDM1 expression levels were significantly reduced. Inhibition of the WNT signal demonstrated the opposite effect. Similarly, inhibitors of BMP and the Nodal/Activin signal were used to determine the effect of signal pathways on differentiation. CER1 affected the Wnt signal and differentiation, but the inhibitor SB only regulated differentiation. BMP-WNT-NODAL were mainly responsible for regulating differentiation. Our results provide a reliable theoretical basis and feasibility for further clinical medical research.

Keywords: Bone morphogenetic protein; Differentiation; Embryonic stem cells; Nodal/Activin; Wnt signalling pathway.

Map-based cloning was used to identify the BrWAX2 gene, which was involved in the cuticular wax biosynthesis. The malfunction of BrWAX2 together with other reduced expression of genes in alkane-forming pathway caused the glossy phenotype. Cuticular wax covering the outer plant surface plays various roles in protecting against biotic and abiotic stresses. Wax-less mutant shows glossy in stem and leaf surface and plays important roles in enriching Chinese cabbage germplasm resources for breeding brilliant green varieties. However, genes responsible for the glossy trait in Chinese cabbage are rarely reported. In this study, we identified a glossy Chinese cabbage line Y1211-1. Genetic analysis indicated that the glossy trait in Y1211-1 was controlled by a single recessive locus, BrWAX2 (Brassica rapa WAX 2). Using bulked segregant sequencing (BSA-Seq) and kompetitive allele-specific PCR (KASP) assays, BrWAX2 was fine-mapped to an interval of 100.78 kb. Functional annotation analysis, expression analysis, and sequence variation analysis revealed that Bra032670, homologous to CER1 in Arabidopsis, was the most likely candidate gene for BrWAX2. The gene Bra032670 was absent in glossy mutant. Cuticular wax composition analysis and RNA-Seq analysis suggested that the absence of BrWAX2 together with the decreased expression of other genes in alkane-forming pathway reduced the wax amount and caused the glossy phenotype. Furthermore, we developed and validated the functional marker BrWAX2-sp for BrWAX2. Overall, these results provide insight into the molecular mechanism underlying cuticular wax biosynthesis and reveal valuable information for marker-assisted selection (MAS) breeding in Chinese cabbage.

The outer epidermal cell walls of plant shoots are covered with a cuticle, a continuous lipid structure that provides protection from desiccation, UV light, pathogens, and insects. The cuticle is mostly composed of cutin and cuticular wax. Cuticular wax synthesis is synchronized with surface area expansion during plant development and is associated with plant responses to biotic and abiotic stresses. Cuticular wax deposition is tightly regulated by well-established transcriptional and post-transcriptional regulatory mechanisms, as well as post-translationally via the ubiquitin-26S proteasome system (UPS). The UPS is highly conserved in eukaryotes and involves the covalent attachment of polyubiquitin chains to the target protein by an E3 ligase, followed by the degradation of the modified protein by the 26S proteasome. A large number of E3 ligases are encoded in the Arabidopsis genome, but only a few have been implicated in the regulation of cuticular wax deposition. In this study, we have conducted an E3 ligase reverse genetic screen and identified a novel RING-type E3 ubiquitin ligase, AtARRE, which negatively regulates wax biosynthesis in Arabidopsis. Arabidopsis plants overexpressing AtARRE exhibit glossy stems and siliques, reduced fertility and fusion between aerial organs. Wax load and wax compositional analyses of AtARRE overexpressors showed that the alkane-forming branch of the wax biosynthetic pathway is affected. Co-expression of AtARRE and candidate target proteins involved in alkane formation in both Nicotiana benthamiana and stable Arabidopsis transgenic lines demonstrated that AtARRE controls the levels of wax biosynthetic enzymes ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3). CER1 has also been confirmed to be a ubiquitination substrate of the AtARRE E3 ligase by an in vivo ubiquitination assay using a reconstituted Escherichia coli system. The AtARRE gene is expressed throughout the plant, with the highest expression detected in fully expanded rosette leaves and oldest stem internodes. AtARRE gene expression can also be induced by exposure to pathogens. These findings reveal that wax biosynthesis in mature plant tissues and in response to pathogen infection is controlled post-translationally.

Keywords: Arabidopsis; AtARRE; CER1; CER3; E3 ligase; cuticle; cuticular wax biosynthesis.

Apoplastic barriers in the endodermis, such as Casparian strips and suberin lamellae, control the passage of water and minerals into the stele. Apoplastic barriers are thus thought to contribute to salt exclusion in salt-excluding plants such as sweet sorghum (Sorghum bicolor). However, little is known about the genes involved in the development of the apoplastic barrier. Here, we identified candidate genes involved in Casparian strip and suberin lamella development in the roots of a sweet sorghum line (M-81E). Three distinct developmental regions (no differentiation, developing, and mature) were identified based on Casparian strip and suberin lamella staining in root cross sections. Sequencing of RNA extracted from these distinct sections identified key genes participating in the differentiation of the apoplastic barrier. The different sections were structurally distinct, presumably due to differences in gene expression. Genes controlling the phenylpropanoid pathway, fatty acid elongation, and fatty acid ω-hydroxylation appeared to be directly responsible for the formation of the apoplastic barrier. Our dataset elucidates the molecular processes underpinning apoplastic barrier development and provides a basis for future research on molecular mechanisms of apoplastic barrier formation and salt exclusion.Abbreviations: SHR, SHORTROOT; MYB, MYB DOMAIN PROTEIN; CIFs, Casparian strip integrity factors; CASP, Casparian strip domain proteins; PER, peroxidase; ESB1, ENHANCED SUBERIN1; CS, Casparian strip; RPKM, reads per kilobase per million reads; DEGs, differentially expressed genes; FDR, false discovery rate; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RNA-seq, RNA sequencing; PAL, phenylalanine ammonia-lyase; CYP, cytochrome P450 monooxygenases; 4CL, 4-coumarate-CoA ligase; AAE5, ACYL-ACTIVATING ENZYME5; CCR, cinnamoyl CoA reductase; TKPR, TETRAKETIDE ALPHA-PYRONE REDUCTASE1; CAD, cinnamyl alcohol dehydrogenase; HST, shikimate O-hydroxycinnamoyltransferase; PMAT2, PHENOLIC GLUCOSIDE MALONYLTRANSFERASE2; CCOAOMT, caffeoyl-CoA O-methyltransferase; KCS, β-ketoacyl-CoA synthase; CUT1, CUTICULAR PROTEIN1; DET2, 5-alpha-reductase; TAX, 3'-N-debenzoyl-2'-deoxytaxol N-benzoyltransferase; CER1, ECERIFERUM1; FAR, fatty acyl reductase; AF-CoA, alcohol-forming fatty acyl-CoA reductase; ABCG, ATP-binding cassette, subfamily G; ERF, ethylene-responsive transcription factor; HSF, heat stress transcription factor; NTF, NUCLEAR TRANSCRIPTION FACTOR Y SUBUNIT B-5; GPAT, glycerol 3-phosphate acyltransferase.

Cuticular waxes play important eco-physiological roles in protecting plants against abiotic and biotic stresses and show high sensitivity to environmental changes. In order to clarify the responses of cuticular waxes on faba bean (Vicia faba L.) leaves to different light wavelengths, the phenotypic plasticity of cuticular waxes was analyzed when plants were subjected to white, red, yellow, blue, and purple light. Leaf samples from yellow, purple, and white lights were further analyzed, and candidate genes of wax biosynthesis were selected by RNA-seq technology and transcriptome processing. Yellow light increased the total wax coverage and changed the crystal structure compared with leaves under white light. Light wavelengths changed the relative abundance of dominant primary alcohol from C₂₄ under white, yellow, and red lights to C₂₆ under blue and purple lights. In total, 100,194 unigenes were obtained, and 10 genes were annotated in wax biosynthesis pathway, including VLCFAs elongation (KCS1, KCS4, LACS2 and LACS9), acyl reduction pathway (FAR3 and WSD1), and decarboxylation pathway (CER1, CER3 and MAH1). qRT-PCR analysis revealed that yellow and purple lights significantly influenced the expression levels of these genes. Yellow light also increased the water loss rate and decreased the photosynthesis rate. Light at different wavelengths particularly yellow light induced the changes of phenotypic plasticity of cuticular waxes, which thus altered the leaf eco-physiological functions. The expression levels of genes related to wax biosynthesis were also altered by different light wavelengths, suggesting that light at different wavelengths may also be applied in selecting candidate genes involved in wax biosynthesis in other crops.

As a barrier for plants to contact with the outside world, epidermal wax plays an important role in resisting biotic and abiotic stresses. In this study, we analyzed the effect of wax content on leaf permeability by measuring the wax loss rate in the leaf. To further clarify the wax composition of the wheat epidermis and its molecular regulation mechanism, we applied untargeted lipidomic and transcriptome analysis on the leaf epidermis wax of Jimai 22 low-wax mutant (waxless) and multi-wax mutant (waxy). Our research showed that the mutant waxy has a slow loss rate, which can maintain higher leaf water content. 31 lipid subclasses and 1,367 lipid molecules were identified. By analyzing the wax differences of the two mutants, we found that the main lipid components of leaf epidermis wax in Jimai 22 were WE (C19-C50), DG (C27-C53), MG (C31-C35), and OAHFA (C31-C52). Carbon chain length analysis showed that, in wheat epidermis wax, WE was dominated by C44 molecules, DG was mainly concentrated in C47, C45, C37, and C31 molecules, C48 played a leading role in OAHFA, and C35 and C31 played a major role in MG. Among them, DG, MG, and OAHFA were detected in wheat leaf wax for the first time, and they were closely related to stress resistance. Compared with the waxy, 6,840 DEGs were detected in the mutant waxless, 3,181 DEGs were upregulated, and 3,659 DEGs were downregulated. The metabolic pattern of main waxy components in the wheat epidermis was constructed according to KEGG metabolic pathway and 46 related genes were screened, including KSC, TER, FAR, WSD1, CER1, MAH1, ALDH7A1, CYP704B1, ACOT1_2_4, CYP86, MGLL, GPAT, ALDH, DPP1, dgkA, plsC, and E2.3.1.158 related genes. The screened wax-related genes were confirmed to be highly reliable by qRT-PCR. In addition, we found TER gene TraesCS6B03G1132900LC in wheat mutant waxless leaves for the first time, which inhibited the synthesis of long-chain acyl-CoA (n+2) by downregulating its expression. These results provide valuable reference information for further study of wheat epidermis wax heredity and molecular regulation.

Keywords: liposome; molecular regulation; transcriptome; wax; wheat.

The cuticular wax of fruit is closely related to quality, storability, and pathogen susceptibility after harvest. However, little is known about the cuticular wax of goji berry (Lycium barbarum L.) cultivars. In the present study, the chemical composition, crystal structures, and expression levels of associated genes of the cuticular wax of six goji cultivars were investigated. We detected 70 epicuticular wax compounds in six goji cultivars. Among them, fatty acids, alkanes, and primary alcohols were the major components of the cuticular wax of goji berries, which were related to the formation of irregular lamellar crystal structures. The terpenoid compounds in the cuticular wax of goji berries were highly resistant to Alternaria rot. Moreover, the CER1, CER6, LACS1, MAH1, LTP4, ABC11, MYB96, and WIN1 genes in goji berries might be closely related to wax synthesis. These results provide valuable information for breeding and screening goji cultivars suitable for postharvest storage.

Keywords: chemical composition; crystal morphology; cuticular wax; gene expression; goji berries.

Objective: The aim of our study was to investigate the relationship between the rs74434454 polymorphism of the CER1 gene and selected biochemical, densitometric and anthropometric markers in Slovak postmenopausal women of two ethnic groups: Roma and non-Roma.

Subjects and methods: The scientific study included 303 postmenopausal women of the non-Roma and Roma populations who were divided into two groups based on densitometric measurements: control group (CG) and osteoporotic group (OG). Genomic DNA was isolated from peripheral blood using a commercial NucleoSpin® Blood kit following a standard protocol. The TaqMan Real-Time PCR method was used for genotyping. Biochemical markers were measured with Cobas e411 and Cobas Integra400 plus analysers.

Results: In the control group of postmenopausal Roma women, the occurrence of the risk genotype GG was not observed. In the group of Roma women with osteopenia and osteoporosis, the GG genotype occurred at a frequency of 3.03%. In the group of non-Roma women (between CG and OG) statistically significant differences were found in all monitored biochemical markers except CTx-I (p<0.66). In contrast, in the group of Roma women, statistical significance was only found in the osteoresorption marker CTx-I (p<0.007). In the population of Roma women, we did not find a statistically significant difference between the AA, AG and GG genotypes in any of the monitored markers.

Conclusions: The results provide the first and unique insight on the distribution of genotypes and alleles of the rs74434454 CER1 gene polymorphism and its relationship to markers of bone metabolism in two ethnically distinct groups.

The chemical composition of a plant cuticle can change in response to various abiotic or biotic stresses and plays essential functions in disease resistance responses. Arabidopsis thaliana mutants altered in cutin content are resistant to Botrytis cinerea, presumably because of increased cuticular water and solute permeability, allowing for faster induction of defense responses. Within this context, our knowledge of wax mutants is limited against this pathogen. We tested the contribution of cuticular components to immunity to B. cinerea using mutants altered in either cutin or wax alone, or in both cutin and wax contents. We found that even all the tested mutants showed increased permeability and reactive oxygen species (ROS) accumulation in comparison with wild-type plants and that only cutin mutants showed resistance. To elucidate the early molecular mechanisms underlying cuticle-related immunity, we performed a transcriptomic analysis. A set of upregulated genes involved in cell wall integrity and accumulation of ROS were shared by the cutin mutants bdg, lacs2-3, and eca2, but not by the wax mutants cer1-4 and cer3-6. Interestingly, these genes have recently been shown to be required in B. cinerea resistance. In contrast, we found the induction of genes involved in abiotic stress shared by the two wax mutants. Our study reveals new insight that the faster recognition of a pathogen by changes in cuticular permeability is not enough to induce resistance to B. cinerea, as has previously been hypothesized. In addition, our data suggest that mutants with resistant phenotype can activate other defense pathways, different from those canonical immune ones.

Keywords: B. cinerea; ROS; cell wall; cuticle; cuticular mutants; permeability.

The epidermal stratum corneum (SC) lipid matrix, principally consisting of an equimolar ratio of ceramides, free fatty acids, and cholesterol, plays a crucial role in maintaining proper skin barrier function. Conditions which impair barrier integrity, such as in atopic dermatitis, correlate with the alternation of key ceramide subclasses and reduced chain length of acyl moieties. However, there is limited knowledge about the impact of unprotected repeat sun exposure on the skin lipid composition, especially ceramide profiles.This study investigated the effects of ultraviolet (UV) radiation on the ceramide profile using both an ex vivo skin and a clinical model. Lipidomic analysis of UV-exposed skin showed shifts to the composition of ceramide subclasses essential in repairing and strengthening the SC barrier (including CER1[EOS], CER3[NP], and CER6[AP]) and reduced very long-chain acyl moieties. Gene expression analysis and immunohistochemical staining of key enzymes (aSMase, DES1, CerS5, CerS3) suggested that lipid alterations can be attributed to changes within the ceramide biosynthesis process. Topical application of ceramide-containing suncare products help maintain SC-essential ceramide subclasses and proper ceramide chain length, demonstrating the importance of proper photoprotection to maintain healthy skin barrier and ceramide quality during daily sun exposure. J Drugs Dermatol. 2022;21(1):77-85. doi:10.36849/JDD.6331.

The DAN gene family (DAN, Differential screening-selected gene Aberrant in Neuroblastoma) is a group of genes that is expressed during development and plays fundamental roles in limb bud formation and digitation, kidney formation and morphogenesis and left-right axis specification. During adulthood the expression of these genes are associated with diseases, including cancer. Although most of the attention to this group of genes has been dedicated to understanding its role in physiology and development, its evolutionary history remains poorly understood. Thus, the goal of this study is to investigate the evolutionary history of the DAN gene family in vertebrates, with the objective of complementing the already abundant physiological information with an evolutionary context. Our results recovered the monophyly of all DAN gene family members and divide them into five main groups. In addition to the well-known DAN genes, our phylogenetic results revealed the presence of two new DAN gene lineages; one is only retained in cephalochordates, whereas the other one (GREM3) was only identified in cartilaginous fish, holostean fish, and coelacanth. According to the phyletic distribution of the genes, the ancestor of gnathostomes possessed a repertoire of eight DAN genes, and during the radiation of the group GREM1, GREM2, SOST, SOSTDC1, and NBL1 were retained in all major groups, whereas, GREM3, CER1, and DAND5 were differentially lost.

Keywords: Cerberus; Differential retention; Evolutionary medicine; Evolutionary slowdown; Gene family evolution; Gremlin.

Very-long-chain (VLC) alkanes are the main wax compounds of tomato fruit and leaf. ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3) are the two key genes involved in VLC alkane biosynthesis in Arabidopsis thaliana. However, the CER1 and CER3 homologous genes in tomato have not been investigated and their exact biological function remains unknown. We analyzed the wax profiles in tomato leaves and fruits at different growth stages, and characterized the CER1 and CER3 homologous genes. VLC alkanes were the predominant wax compounds both in the leaf and fruit at all developmental stages. We identified five CER1 homologs and two CER3 homologs in tomato, which were designated as SlCER1-1 to SlCER1-5 and SlCER3-1 and SlCER3-2 respectively. The genes exhibited tissue- and organ-dependent expression patterns and were induced by abiotic stresses. SlCER1-1 was localized to the endoplasmic reticulum (ER), which is also the main site of wax biosynthesis. Silencing the SlCER1-1 gene in tomato significantly reduced the amounts of n-Alkanes and branched alkanes, whereas its overexpression in Arabidopsis had the opposite effect. Under drought stress, both n-Alkanes and branched alkanes increased significantly in wild-type but not the SlCER1-1 RNAi tomato plants. Furthermore, SlCER1-1 silencing also increased the cuticular permeabilities of the leaves and fruits. In conclusion, SlCER1-1 is involved in wax alkane biosynthesis in tomato and plays an important role in the drought tolerance and fruit storability.