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In flowering plants, repression of the seed maturation program is essential for the transition from the seed to the vegetative phase, but the underlying mechanisms remain poorly understood. The B3-domain protein VIVIPAROUS1/ABSCISIC ACID-INSENSITIVE3-LIKE 1 (VAL1) is involved in repressing the seed maturation program. Here we uncovered a molecular network triggered by the plant hormone brassinosteroid (BR) that inhibits the seed maturation program during the seed-to-seedling transition in Arabidopsis (Arabidopsis thaliana). val1-2 mutant seedlings treated with a BR biosynthesis inhibitor form embryonic structures, whereas BR signaling gain-of-function mutations rescue the embryonic structure trait. Furthermore, the BR-activated transcription factors BRI1-EMS-SUPPRESSOR 1 and BRASSINAZOLE-RESISTANT 1 bind directly to the promoter of AGAMOUS-LIKE15 (AGL15), which encodes a transcription factor involved in activating the seed maturation program, and suppress its expression. Genetic analysis indicated that BR signaling is epistatic to AGL15 and represses the seed maturation program by downregulating AGL15. Finally, we showed that the BR-mediated pathway functions synergistically with the VAL1/2-mediated pathway to ensure the full repression of the seed maturation program. Together, our work uncovered a mechanism underlying the suppression of the seed maturation program, shedding light on how BR promotes seedling growth.

Seed dormancy is an adaptive trait that is crucial to plant survival. Abscisic acid (ABA) is the primary phytohormone that induces seed dormancy. However, little is known about how the level of ABA in seeds is determined. Here we show that the Arabidopsis (Arabidopsis thaliana) H3K27me3 demethylase RELATIVE OF EARLY FLOWERING6 (REF6) suppresses seed dormancy by inducing ABA catabolism in seeds. Seeds of the ref6 loss-of-function mutants displayed enhanced dormancy that was associated with increased endogenous ABA content. We further show that the transcripts of two genes key to ABA catabolism, CYP707A1 and CYP707A3, but not genes involved in ABA biosynthesis, were significantly reduced in ref6 mutants during seed development and germination. In developing siliques, REF6 bound directly to CYP707A1 and CYP707A3, and was responsible for reducing their H3K27me3 levels. Genetic analysis demonstrated that the enhanced seed dormancy and ABA concentration in ref6 depended mainly on the reduced expression of CYP707A1 and CYP707A3. Conversely, overexpression of CYP707A1 could offset the enhanced seed dormancy of ref6. Taken together, our results revealed an epigenetic regulation mechanism that is involved in the regulation of ABA content in seeds.

Switch defective/sucrose non-fermentable (SWI/SNF) chromatin remodelling complexes are multi-protein machineries that control gene expression by regulating chromatin structure in eukaryotes. However, the full subunit composition of SWI/SNF complexes in plants remains unclear. Here we report that in Arabidopsis thaliana, two homologous glioma tumour suppressor candidate region domain-containing proteins, named BRAHMA-interacting proteins 1 (BRIP1) and BRIP2, are core subunits of plant SWI/SNF complexes. brip1 brip2 double mutants exhibit developmental phenotypes and a transcriptome remarkably similar to those of BRAHMA (BRM) mutants. Genetic interaction tests indicated that BRIP1 and BRIP2 act together with BRM to regulate gene expression. Furthermore, BRIP1 and BRIP2 physically interact with BRM-containing SWI/SNF complexes and extensively co-localize with BRM on chromatin. Simultaneous mutation of BRIP1 and BRIP2 results in decreased BRM occupancy at almost all BRM target loci and substantially reduced abundance of the SWI/SNF assemblies. Together, our work identifies new core subunits of BRM-containing SWI/SNF complexes in plants and uncovers the essential role of these subunits in maintaining the abundance of SWI/SNF complexes in plants. The Arabidopsis SWI/SNF complexes regulate chromatin structure and gene expression, and contain many subunits, including BRAHMA. Two homologous BRAHMA-interacting proteins, namely BRIP1 and BRIP2, are identified as new core subunits of the SWI/SNF complex.

Seed is vital to the conservation of germplasm and plant biodiversity. Seed dormancy is an adaptive trait in numerous seed-plant species, enabling plants to survive under stressful conditions. Seed dormancy is mainly controlled by abscisic acid (ABA) and gibberellin (GA) and can be classified as primary and secondary seed dormancy. The primary seed dormancy is induced by maternal ABA. Here we found that AtPER1, a seed-specific peroxiredoxin, is involved in enhancing primary seed dormancy. Two loss-of-function atper1 mutants, atper1-1 and atper1-2, displayed suppressed primary seed dormancy accompanied with reduced ABA and increased GA contents in seeds. Furthermore, atper1 mutant seeds were insensitive to abiotic stresses during seed germination. The expression of several ABA catabolism genes (CYP707A1, CYP707A2, and CYP707A3) and GA biosynthesis genes (GA20ox1, GA20ox3, and KAO3) in atper1 mutant seeds was increased compared to wild-type seeds. The suppressed primary seed dormancy of atper1-1 was completely reduced by deletion of CYP707A genes. Furthermore, loss-of-function of AtPER1 cannot enhance the seed germination ratio of aba2-1 or ga1-t, suggesting that AtPER1-enhanced primary seed dormancy is dependent on ABA and GA. Additionally, the level of reactive oxygen species (ROS) in atper1 mutant seeds was significantly higher than that in wild-type seeds. Taken together, our results demonstrate that AtPER1 eliminates ROS to suppress ABA catabolism and GA biosynthesis, and thus improves the primary seed dormancy and make the seeds less sensitive to adverse environmental conditions.

PurposeFacioscapulohumeral muscular dystrophy (FSHD) is a common adult muscular dystrophy. Over 95% of FSHD cases are associated with contraction of the D4Z4 tandem repeat (~3.3 kb per unit) at 4q35 with a specific genomic configuration (haplotype) called 4qA. Molecular diagnosis of FSHD typically requires pulsed-field gel electrophoresis with Southern blotting. We aim to develop novel genomic and computational methods for characterising D4Z4 repeat numbers in FSHD.MethodsWe leveraged a single-molecule optical mapping platform that maps locations of restriction enzyme sites on high molecular weight (>150 kb) DNA molecules. We developed bioinformatics methods to address several challenges, including the differentiation of 4qA with 4qB alleles, the differentiation of 4q35 and 10q26 segmental duplications, the quantification of repeat numbers with different enzymes that may or may not have recognition sites within D4Z4 repeats. We evaluated the method on 25 human subjects (13 patients, 3 individual control subjects, 9 control subjects from 3 families) labelled by the Nb.BssSI and/or Nt.BspQI enzymes.ResultsWe demonstrated that the method gave a direct quantitative measurement of repeat numbers on D4Z4 repeats with 4qA allelic configuration and the levels of postzygotic mosaicism. Our method had high concordance with Southern blots from several cohorts on two platforms (Bionano Saphyr and Bionano Irys), but with improved quantification of repeat numbers.ConclusionWhile the study is limited by small sample size, our results demonstrated that single-molecule optical mapping is a viable approach for more refined analysis on genotype-phenotype relationships in FSHD, especially when postzygotic mosaicism is present.

SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

Both Histone Deacetylases HDA6 and HDA9 belong to class I subfamily of RPD3/HDA1 HDACs. Loss-of-function mutants of HDA9 form slightly blunt siliques. However, the involvement of HDA6 in regulating silique tip growth is unclear. In this study, we show that HDA6 acts redundantly with HDA9 in regulating the elongation of valve cells in the silique tip. Although the hda6 single mutant does not exhibit a detectable silique phenotype, the silique tip of hda6 hda9 double mutant displays a more severe bulge, a morphology we termed as “nock-shaped”. The valve cells of the silique tip of hda9 are longer than wild-type, and loss of HDA6 in hda9 enhances the valve cell elongation phenotype. The transcript levels of auxin-signaling-related genes are mis-regulated in hda9 and hda6 hda9 siliques, and the GFP reporter driven by the auxin response promoter DR5 is weaker in hda9 or hda6 hda9 than wild-type or hda6. Thus, our findings reveal that HDA6 and HDA9 coordinately control the elongation of silique valve cells through regulating the expression of auxin-related genes in silique tips.

Additional file of Analysis of common bean expressed sequence tags identifies sulfur metabolic pathways active in seed and sulfur-rich proteins highly expressed in the absence of phaseolin and major lectins

Clinical genetic testing results are compiled into a standardized report by genetic specialists and provided to clinicians and patients (Should the patient be intellectually disabled or under 18, the report will be provided to his/her parents or legal guardians). The content of genetic testing report should conform to relevant guidelines, industry standards and consensus. The decisions of clinicians will be made based on the report and clinical indications. Genetic counselors should provide post-test counseling to clinicians and patients or their authorized family members. A mechanism of follow-up visit after the genetic testing should be established with informed consent. Data should be shared by clinical institutions and genome sequencing institutions. As findings upon follow-up visit can help with further evaluation of the results, genome sequencing institutions should regularly re-analyze historical and follow-up data, and the updated results should be shared with clinical institutions. All activities involving reporting, genetic counselling, follow-up visiting, and re-analyzing should follow the relevant guidelines and regulations.

Pre-testing preparation is the basis and starting point of genetic testing. The process includes collection of clinical information, formulation of testing scheme, genetic counseling before testing, and completion of informed consent and testing authorization. To effectively identify genetic diseases in clinics can greatly improve the diagnostic rate of next generation sequencing (NGS), thereby reducing medical cost and improving clinical efficacy. The analysis of NGS results relies, to a large extent, on the understanding of genotype-phenotype correlations, therefore it is particularly important to collect and evaluate clinical phenotypes and describe them in uniform standard terms. Different types of genetic diseases or mutations may require specific testing techniques, which can yield twice the result with half the effort. Pre-testing genetic counseling can help patients and their families to understand the significance of relevant genetic testing, formulate individualized testing strategies, and lay a foundation for follow-up.

Phenylketonuria (PKU) is an autosomal recessive hereditary disease and a common disorder of amino acid metabolism. The average incidence of PKU in China is approximately 1/11 000. It is characterized by lower incidence in the South and higher incidence in the North, particularly the Northwest. PKU is a treatable disease and has been listed in the national newborn screening program. Neonates with positive indication of screening can achieve satisfactory therapeutic effect by timely control of phenylalanine intake after the definite diagnosis. This guideline aims to summarize the knowledge of medical genetics and key points of clinical management of PKU, so as to improve the diagnostic level and standardize newborn screening and clinical treatment of patients.

Bioinformatic analysis and variant classification are the key components of high-throughput sequencing-based genetic diagnostic approach. This consensus is part of the effort to develop a standardized process for next generation sequencing (NGS)-based test for germline mutations underlying Mendelian disorders in China. The flow-chart, common software, key parameters of bioinformatics pipeline for data processing, annotation, storage and variant classification are reviewed, which is aimed to help improving and maintaining a high-quality process and obtaining consistent outcomes for NGS-based molecular diagnosis.

With high accuracy and precision, next generation sequencing (NGS) has provided a powerful tool for clinical testing of genetic diseases. To follow a standardized experimental procedure is the prerequisite to obtain stable, reliable, and effective NGS data for the assistance of diagnosis and/or screening of genetic diseases. At a conference of genetic testing industry held in Shanghai, May 2019, physicians engaged in the diagnosis and treatment of genetic diseases, experts engaged in clinical laboratory testing of genetic diseases and experts from third-party genetic testing companies have fully discussed the standardization of NGS procedures for the testing of genetic diseases. Experts from different backgrounds have provided opinions for the operation and implementation of NGS testing procedures including sample collection, reception, preservation, library construction, sequencing and data quality control. Based on the discussion, a consensus on the standardization of the testing procedures in NGS laboratories is developed with the aim to standardize NGS testing and accelerate implementation of NGS in clinical settings across China.

Additional file 2:Percent of ESTs assigned to a Gene Ontology category per developmental stage (%). (PDF 36 KB)

Additional file 1:Free amino acid profiles in developing seeds of BAT93. (PDF 49 KB)

Background Banana (Musa spp.) is one of the world’s most important fruits and its production is largely limited by diverse stress conditions. SROs (SIMILAR TO RCD-ONE) have important functions in abiotic stress resistance and development of plants. They contain a catalytic core of the poly(ADP-ribose) polymerase (PARP) domain and a C-terminal RST (RCD-SRO-TAF4) domain. In addition, partial SROs also include an N-terminal WWE domain. Although a few of SROs have been characterized in some model plants, little is known about their functions in banana, especially in response to biotic stress. Results Six MaSRO genes in banana genome were identified using the PARP and RST models as a query. Phylogenetic analysis showed that 77 SROs from 15 species were divided into two structurally distinct groups. The SROs in the group I possessed three central regions of the WWE, PARP and RST domains. The WWE domain was lacking in the group II SROs. In the selected monocots, only MaSROs of banana were present in the group II. Most of MaSROs expressed in more than one banana tissue. The stress- and hormone-related cis-regulatory elements (CREs) in the promoter regions of MaSROs supported differential transcripts of MaSROs in banana roots treated with abiotic and biotic stresses. Moreover, expression profiles of MaSROs in the group I were clearly distinct with those observed in the group II after hormone treatment. Notably, the expression of MaSRO4 was significantly upregulated by the multiple stresses and hormones. The MaSRO4 protein could directly interact with MaNAC6 and MaMYB4, and the PARP domain was required for the protein-protein interaction. Conclusions Six MaSROs in banana genome were divided into two main groups based on the characteristics of conserved domains. Comprehensive expression analysis indicated that MaSROs had positive responses to biotic and abiotic stresses via a complex interaction network with hormones. MaSRO4 could interact directly with MaNAC6 and MaMYB4 through the PARP domain to regulate downstream signaling pathway. Electronic supplementary material The online version of this article (10.1186/s12870-019-1807-x) contains supplementary material, which is available to authorized users.

How plants can distinguish pathogenic and symbiotic fungi remains largely unknown. Here, we characterized the role of MaLYK1, a lysin motif receptor kinase of banana. Live cell imaging techniques were used in localization studies. RNA interference (RNAi)-silenced transgenic banana plants were generated to analyze the biological role of MaLYK1. The MaLYK1 ectodomain, chitin beads, chitooligosaccharides (COs) and mycorrhizal lipochitooligosaccharides (Myc-LCOs) were used in pulldown assays. Ligand-induced MaLYK1 complex formation was tested in immunoprecipitation experiments. Chimeric receptors were expressed in Lotus japonicus to characterize the function of the MaLYK1 kinase domain. MaLYK1 was localized to the plasma membrane. MaLYK1 expression was induced by Foc4 (Fusarium oxysporum f. sp. cubense race 4) and diverse microbe-associated molecular patterns. MaLYK1-silenced banana lines showed reduced chitin-triggered defense responses, increased Foc4-induced disease symptoms and reduced mycorrhization. The MaLYK1 ectodomain was pulled down by chitin beads and LCOs or COs impaired this process. Ligand treatments induced MaLYK1 complex formation in planta. The kinase domain of MaLYK1 could functionally replace that of the chitin elicitor receptor kinase 1 (AtCERK1) in Arabidopsis thaliana and of a rhizobial LCO (Nod factor) receptor (LjNFR1) in L. japonicus. MaLYK1 represents a central molecular switch that controls defense- and symbiosis-related signaling.

Seed longevity, the maintenance of viability during storage, is a major factor for conservation of genetic resources and biodiversity. Seed longevity is an important trait of agriculture crop and is impaired by reactive oxygen species (ROS) during seed desiccation, storage and germination (C. R. Biol., 331, 2008 and 796). Seeds possess a wide range of systems (protection, detoxification, repair) allowing them to survive during storage and to preserve a high germination ability. In many plants, 1-cys peroxiredoxin (1-Cys Prx, also named PER1) is a seed-specific antioxidant which eliminates ROS with cysteine residues. Here we identified and characterized a seed-specific PER1 protein from seeds of sacred lotus (Nelumbo nucifera Gaertn.). Purified NnPER1 protein protects DNA against the cleavage by ROS in the mixed-function oxidation system. The transcription and protein accumulation of NnPER1 increased during seed desiccation and imbibition and under abiotic stress treatment. Ectopic expression of NnPER1 in Arabidopsis enhanced the seed germination ability after controlled deterioration treatment (CDT), indicating that NnPER1 improves seed longevity of transgenic plants. Consistent with the function of NnPER1 on detoxifying ROS, we found that the level of ROS release and lipid peroxidation was strikingly lower in transgenic seeds compared to wild-type with or without CDT. Furthermore, transgenic Arabidopsis seeds ectopic-expressing NnPER1 displayed enhanced tolerance to high temperature and abscisic acid (ABA), indicating that NnPER1 may participate in the thermotolerance and ABA signaling pathway.

SWI/SNF-type chromatin remodelers, such as BRAHMA (BRM), and H3K27 demethylases both have active roles in regulating gene expression at the chromatin level1–5, but how they are recruited to specific genomic sites remains largely unknown. Here we show that RELATIVE OF EARLY FLOWERING 6 (REF6), a plant-unique H3K27 demethylase6, targets genomic loci containing a CTCTGYTY motif via its zinc-finger (ZnF) domains and facilitates the recruitment of BRM. Genome-wide analyses showed that REF6 colocalizes with BRM at many genomic sites with the CTCTGYTY motif. Loss of REF6 results in decreased BRM occupancy at BRM–REF6 co-targets. Furthermore, REF6 directly binds to the CTCTGYTY motif in vitro, and deletion of the motif from a target gene renders it inaccessible to REF6 in vivo. Finally, we show that, when its ZnF domains are deleted, REF6 loses its genomic targeting ability. Thus, our work identifies a new genomic targeting mechanism for an H3K27 demethylase and demonstrates its key role in recruiting the BRM chromatin remodeler.

SummarySPT6 is a conserved transcription regulator that is generally viewed as an elongation factor. However, emerging evidence show its potential role in the control of transcription initiation at genic and intragenic promoters. Here we first present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNA Polymerase II (RNAPII) occupancy across transcribed genes. Further, we show that SPT6L enrichment is shifted, unexpectedly, from gene body to the transcription starting site (TSS) when its association with RNAPII is disrupted. Finally, we demonstrate that recruitment of SPT6L starts at TSS, and then spreads to the gene body during transcription. These findings refine the mechanisms underlying SPT6L recruitment in transcription and shed light on the role of SPT6L in transcription initiation.

Facioscapulohumeral Muscular Dystrophy (FSHD) is a common adult muscular dystrophy in which the muscles of the face, shoulder blades and upper arms are among the most affected. FSHD is the only disease in which “junk” DNA is reactivated to cause disease, and the only known repeat array-related disease where fewer repeats cause disease. More than 95% of FSHD cases are associated with copy number loss of a 3.3kb tandem repeat (D4Z4 repeat) at the subtelomeric chromosomal region 4q35, of which the pathogenic allele contains less than 10 repeats and has a specific genomic configuration called 4qA. Currently, genetic diagnosis of FSHD requires pulsed-field gel electrophoresis followed by Southern blot, which is labor-intensive, semi-quantitative and requires long turnaround time. Here, we developed a novel approach for genetic diagnosis of FSHD, by leveraging Bionano Saphyr single-molecule optical mapping platform. Using a bioinformatics pipeline developed for this assay, we found that the method gives direct quantitative measurement of repeat numbers, can differentiate 4q35 and the highly paralogous 10q26 regions, can determine the 4qA/4qB allelic configuration, and can quantitate levels of post-zygotic mosaicism. We evaluated this approach on 5 patients (including two with post-zygotic mosaicism) and 2 patients (including one with post-zygotic mosaicism) from two separate cohorts, and had complete concordance with Southern blots, but with improved quantification of repeat numbers resolved between haplotypes. We concluded that single-molecule optical mapping is a viable approach for molecular diagnosis of FSHD and may be applied in clinical diagnostic settings once more validations are performed.

The widespread application of next generation sequencing (NGS) in clinical settings has enabled testing, diagnosis, treatment and prevention of genetic diseases. However, many issues have arisen in the meanwhile. One of the most pressing issues is the lack of standards for reporting genetic test results across different service providers. The First Forum on Standards and Specifications for Clinical Genetic Testing was held to address the issue in Shenzhen, China, on October 28, 2017. Participants, including geneticists, clinicians, and representatives of genetic testing service providers, discussed problems of clinical genetic testing services across in China and shared opinions on principles, challenges, and standards for reporting clinical genetic test results. Here we summarize expert opinions presented at the seminar and report the consensus, which will serve as a basis for the development of standards and guidelines for reporting of clinical genetic testing results, in order to promote the standardization and regulation of genetic testing services in China.

Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism, eliminates premature termination codon-containing (PTC⁺) transcripts. For instance, it maintains the homeostasis of splicing factors and degrades aberrant transcripts of human genetic disease genes. Here we examine the inhibitory effect on the NMD pathway and consequent increase of PTC+ transcripts by the dietary compound curcumin. We have found that several PTC⁺ transcripts including that of serine/arginine-rich splicing factor 1 (SRSF1) were specifically increased in cells by curcumin. We also observed a similar curcumin effect on the PTC⁺ mutant transcript from a Tay-Sachs-causing HEXA allele or from a beta-globin reporter gene. The curcumin effect was accompanied by significantly reduced expression of the NMD factors UPF1, 2, 3A and 3B. Consistently, in chromatin immunoprecipitation assays, curcumin specifically reduced the occupancy of acetyl-histone H3 and RNA polymerase II at the promoter region (-376 to -247nt) of human UPF1, in a time- and dosage-dependent way. Importantly, knocking down UPF1 abolished or substantially reduced the difference of PTC(+) transcript levels between control and curcumin-treated cells. The disrupted curcumin effect was efficiently rescued by expression of exogenous Myc-UPF1 in the knockdown cells. Together, our data demonstrate that a group of PTC⁺ transcripts are stabilized by a dietary compound curcumin through the inhibition of UPF factor expression and the NMD pathway.

Background Mutations of PKD1 and PKD2 accounted for the most cases of autosomal dominant polycystic kidney disease (ADPKD). The presence of the large transcript, numerous exons and complex reiterated regions within the gene has significantly complicated the analysis of PKD1 with routine PCR-based approaches. Methods We developed a strategy to analyze both the PKD1/PKD2 genes simultaneously using targeted next-generation sequencing (NGS). All coding exons plus the flanking sequences of PKD1 and PKD2 genes from probands were captured, individually barcoded and followed by HiSeq2000 sequencing. The candidate variants were validated by using classic Sanger sequencing. PKD1-specific primers were designed to amplify the replicated areas of PKD1 gene. Results Five novel variations and one known mutation in PKD1 gene were detected in five familial and one sporadic Chinese ADPKD patients. Through pedigree and bioinformatic analysis, five of them were identified as pathogenic mutations (p.G1319R, p.Y3781*, p.W4122*, p.Val700Glyfs*14, and p.Leu3656Trpfs*28) and one was as polymorphism (p.T2420I). Conclusions Our result showed that targeted capture and NGS technology were effective for the gene testing of ADPKD disorder. Mutation study of PKD1 and PKD2 genes in Chinese patients may contribute to better understanding of the genetic diversity between different ethnic groups and enrich the mutation database in Asian population.

The seed maturation genes are specifically and highly expressed during late embryogenesis. In this work, yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays revealed that HISTONE DEACETYLASE19 (HDA19) interacted with the HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2-LIKE1 (HSL1), and the zinc-finger CW [conserved Cys (C) and Trp (W) residues] domain of HSL1 was responsible for the interaction. Furthermore, we found that mutations in HDA19 resulted in the ectopic expression of seed maturation genes in seedlings, which was associated with increased levels of gene activation marks, such as Histone H3 acetylation (H3ac), Histone H4 acetylation (H4ac), and Histone H3 Lys 4 tri-methylation (H3K4me3), but decreased levels of the gene repression mark Histone H3 Lys 27 tri-methylation (H3K27me3) in the promoter and/or coding regions. In addition, elevated transcription of certain seed maturation genes was also found in the hsl1 mutant seedlings, which was also accompanied by the enrichment of gene activation marks but decreased levels of the gene repression mark. Chromatin immunoprecipitation assays showed that HDA19 could directly bind to the chromatin of the seed maturation genes. These results suggest that HDA19 and HSL1 may act together to repress seed maturation gene expression during germination. Further genetic analyses revealed that the homozygous hsl1 hda19 double mutants are embryonic lethal, suggesting that HDA19 and HSL1 may play a vital role during embryogenesis.

The dehydration-responsive element binding proteins/C-repeat binding factors (i.e., DREBs/CRT) are crucial transcription factors, they can bind the cis-elements containing the A/GCCGAC sequence in the promoter region. Overexpression of DREB genes enhances the resistance to multiple abiotic stresses, but also causes dwarfism and delayed flowering in many plant species. In this study, constitutive overexpression of AtDREB1A in soybean plants caused dwarfism and delayed-flowering phenotypes. The delayed-flowering phenotype was not affected by day length and could not be reversed by exogenous gibberellic acid. The expression levels of flowering-related genes were determined by quantitative real-time RCR. The Glyma11g13220 (designated as GmVRN1-like) expression was strongly induced in transgenic plants. GmVRN1-like was homologous to the Arabidopsis thaliana VRN1 gene (At3g18990). Electrophoretic mobility shift assay showed that AtDREB1A could bind the dehydration-responsive element motif, ACCGAC, in a region −157 to −186 bp upstream of GmVRN1-like. The motif shared 100 % identity with the DRE sequence present in RD29A. Our results imply that the delayed-flowering of AtDREB1A-overexpressing plants might be caused by up-regulating of GmVRN1-like gene.

Acetyl-coenzyme A (acetyl-CoA) is a central metabolite and the acetyl source for protein acetylation, particularly histone acetylation that promotes gene expression. However, the effect of acetyl-CoA levels on histone acetylation status in plants remains unknown. Here, we show that malfunctioned cytosolic acetyl-CoA carboxylase1 (ACC1) in Arabidopsis leads to elevated levels of acetyl-CoA and promotes histone hyperacetylation predominantly at lysine 27 of histone H3 (H3K27). The increase of H3K27 acetylation (H3K27ac) is dependent on adenosine triphosphate (ATP)-citrate lyase which cleaves citrate to acetyl-CoA in the cytoplasm, and requires histone acetyltransferase GCN5. A comprehensive analysis of the transcriptome and metabolome in combination with the genome-wide H3K27ac profiles of acc1 mutants demonstrate the dynamic changes in H3K27ac, gene transcripts and metabolites occurring in the cell by the increased levels of acetyl-CoA. This study suggests that H3K27ac is an important link between cytosolic acetyl-CoA level and gene expression in response to the dynamic metabolic environments in plants.

A series of genetically related lines of common bean (Phaseolus vulgaris L.) integrate a progressive deficiency in major storage proteins, the 7S globulin phaseolin and lectins. SARC1 integrates a lectin-like protein, arcelin-1 from a wild common bean accession. SMARC1N-PN1 is deficient in major lectins, including erythroagglutinating phytohemagglutinin (PHA-E) but not α-amylase inhibitor, and incorporates also a deficiency in phaseolin. SMARC1-PN1 is intermediate and shares the phaseolin deficiency. Sanilac is the parental background. To understand the genomic basis for variations in protein profiles previously determined by proteomics, the genotypes were submitted to short-fragment genome sequencing using an Illumina HiSeq 2000/2500 platform. Reads were aligned to reference sequences and subjected to de novo assembly. The results of the analyses identified polymorphisms responsible for the lack of specific storage proteins, as well as those associated with large differences in storage protein expression. SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene. While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter. Among residual lectins compensating for storage protein deficiency, mannose lectin FRIL and α-amylase inhibitor 1 genes are uniquely present in SMARC1N-PN1. An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1. An approximately 7-fold increase in accumulation of 11S globulin legumin is not associated with variation in proximal promoter sequence, suggesting that the identity of individual proteins involved in proteome rebalancing might also be determined at the translational level.

Reactive oxygen species (ROS) are toxic by-products generated continuously during seed desiccation, storage, and germination, resulting in seed deterioration and therefore decreased seed longevity. The toxicity of ROS is due to their indiscriminate reactivity with almost any constituent of the cell, such as lipids, proteins, and DNA. The damage to the genome induced by ROS has been recognized as an important cause of seed deterioration. A prominent DNA lesion induced by ROS is 7,8-dihydro-8-oxoguanine (8-oxo-G), which can form base pairs with adenine instead of cytosine during DNA replication and leads to GC→TA transversions. In Arabidopsis, AtOGG1 is a DNA glycosylase/apurinic/apyrimidinic (AP) lyase that is involved in base excision repair for eliminating 8-oxo-G from DNA. In this study, the functions of AtOGG1 were elaborated. The transcript of AtOGG1 was detected in seeds, and it was strongly up-regulated during seed desiccation and imbibition. Analysis of transformed Arabidopsis protoplasts demonstrated that AtOGG1-yellow fluorescent protein fusion protein localized to the nucleus. Overexpression of AtOGG1 in Arabidopsis enhanced seed resistance to controlled deterioration treatment. In addition, the content of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in transgenic seeds was reduced compared to wild-type seeds, indicating a DNA damage-repair function of AtOGG1 in vivo. Furthermore, transgenic seeds exhibited increased germination ability under abiotic stresses such as methyl viologen, NaCl, mannitol, and high temperatures. Taken together, our results demonstrated that overexpression of AtOGG1 in Arabidopsis enhances seed longevity and abiotic stress tolerance.

This study examined the association between 49 markers in the Runt-related transcription factor 2 (RUNX2) gene and nonsyndromic cleft lip with/without cleft palate (CL/P) among 326 Chinese case-parent trios, while considering gene-environment (GxE) interaction and parent-of-origin effects. Five single-nucleotide polymorphisms (SNPs) showed significant evidence of linkage and association with CL/P and these results were replicated in an independent European sample of 825 case-parent trios. We also report compelling evidence for interaction between markers in RUNX2 and environmental tobacco smoke (ETS). Although most marginal SNP effects (i.e., ignoring maternal exposures) were not statistically significant, eight SNPs were significant when considering possible interaction with ETS when testing for gene (G) and GxE interaction simultaneously or when considering GxE alone. Independent samples from European populations showed consistent evidence of significant GxETS interaction at two SNPs (rs6904353 and rs7748231). Our results suggest genetic variation in RUNX2 may influence susceptibility to CL/P through interacting with ETS.

Annexins are multifunctional proteins characterized by their capacity to bind calcium ions and negatively charged lipids. Although there is increasing evidence implicating their importance in plant stress responses, their functions in seeds remain to be further studied. In this study, we identified a heat-induced annexin, NnANN1, from the embryonic axes of sacred lotus (Nelumbo nucifera Gaertn.) using comparative proteomics approach. Moreover, the expression of NnANN1 increased considerably in response to high-temperature treatment. Quantitative real-time PCR (qRT-PCR) revealed that the transcripts of NnANN1 were detected predominantly during seed development and germination in sacred lotus, implicating a role for NnANN1 in plant seeds. Ectopic expression of NnANN1 in Arabidopsis resulted in enhanced tolerance to heat stress in transgenic seeds. In addition, compared to the wild-type seeds, transgenic seeds ectopically expressing NnANN1 exhibited improved resistance to accelerated aging treatment used for assessing seed vigor. Furthermore, transgenic seeds showed enhanced peroxidase activities, accompanied with reduced lipid peroxidation and reduced ROS release levels compared to the wild-type seeds. Taken together, these results indicate that NnANN1 plays an important role in seed thermotolerance and germination vigor.

In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.

Nucleosome loss and histone modifications are important mechanisms for transcriptional regulation. Concomitant changes in chromatin structures of two peanut (Arachis hypogaea L.) oleosin genes, AhOleo17.8 and AhOleo18.5, were examined in relation to transcriptional activity. Spatial and temporal expression analyses showed that both AhOleo17.8 and AhOleo18.5 promoters can adopt three conformational states, an inactive state (in vegetative tissues), a basal activated state (in early maturation embryos), and a fully activated state (in late maturation embryos). Chromatin immunoprecipitation assays revealed an increase of histone H3 acetylation levels at the proximal promoters and coding regions of AhOleo17.8 and AhOleo18.5 associated with basal transcription in early maturation embryos. Meanwhile, a decrease of histone H3K9 dimethylation levels at coding regions of oleosins was observed in early maturation embryos. However, a dramatic decrease in the histone acetylation signal was observed at the core promoters and the coding regions of the two oleosins in the fully activated condition in late maturation embryos. Although a small decrease of histone H3 levels of oleosins chromatin was detected in early maturation embryos, a significant loss of histone H3 levels occurred in late maturation embryos. These analyses indicate that the histone eviction from the proximal promoters and coding regions is associated with the high expression of oleosin genes during late embryos maturation. Moreover, the basal expression of oleosins in early maturation embryos is accompanied by the increase of histone H3 acetylation and decrease of histone H3K9me2.

Aims: Our purpose was to identify the genetic defect in a Chinese cerulean cataract family. Methods: After obtaining informed consent, genomic DNA was extracted from leukocytes. Genotyping and 2-point linkage analysis were carried out using the MLINK component of the LINKAGE program package version 5.10. Mutational analysis of the candidate gene was performed by bidirectional sequencing. The structure homology modeling of the mutant protein was based on Swiss-Model Serve, and its structure was displayed and compared with native β-B2-crystallin using the RasMol software. Results: The disease locus was mapped within a 4.05-cM interval on 22q11.22–22q12.1. By sequencing, a cytosine to thymine transition (NM_000496.2:c.463C>T) was detected in exon 6 of CRYBB2, which resulted in the insertion of a premature stop codon (p.Q155X). In addition, there existed another transition (NM_000496.2:c.471C>T) in the same exon, which does not change the amino acid sequence. Neither NM_000496.2:c.463C>T nor NM_000496.2:c.471C>T were found in 171 normal Chinese controls. The homology modeling showed that the second structure of the mutant protein was different from that of native human β-B2-crystallin. Conclusions: This is the first report of congenital cerulean cataract associated with a mutation in CRYBB2 in a Chinese family. This finding further strengthens the association between CRYBB2 and cerulean cataracts. Two transitions, NM_000496.2:c.463C>T and NM_000496.2:c.471C>T, in CRYBB2 are identical to the sequence of CRYBB2P1, which has over 97% homology to CRYBB2. This supports the possibility of gene conversion between CRYBB2 and CRYBB2P1 as a mechanism responsible for congenital cataract.

In this study, inter-simple sequence repeats (ISSR) markers were applied to assess genetic diversity and genetic relationships of 92 cultivars of sacred lotus (Nelumbo nucifera Gaertn.), one of the most famous flowers in China. Our results showed that sacred lotus exhibited a low level of genetic diversity (percentage of polymorphic bands, PPB = 55.8%), which may result from its asexual mode of reproduction and long-term artificial selection. Clustering analyses indicated that these cultivars could be divided into two clades. Most cultivars of Chinese lotus species origin were included in one clade, and one cultivar of American lotus species origin was nested in the other clade. The hybrid cultivars from hybridization between the two subspecies were interspersed in these two clades. Seven cultivars native to Thailand formed a distinct subclade among the cultivars of Chinese lotus species origin. Genetic differentiation between two subspecies, and between cultivars from Thailand and other cultivars could be attributed to geographic isolation. The monophyly of three cultivars of Sanshui Winter Lotus and their closest relationships to Chinese lotus species origin suggests that they might have a common origin and may consist completely or mainly of genetic material from N. nucifera subsp. nucifera.

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut.

Survival of motor neuron 2 (SMN2) is a modifier gene for spinal muscular atrophy (SMA), a neurodegenerative disease caused by insufficient SMN protein mostly due to SMN1 defect. SMN2 is nearly identical to SMN1 but unfortunately only able to produce a small amount of SMN protein due to exon 7 skipping. The exon 7-containing SMN2 transcript (SMN2_E7+) can be increased by a dietary compound, curcumin, but the involved molecular changes are not clear. Here we have found that in fibroblast cells of a SMA type II patient, curcumin enhanced the inclusion of SMN2 exon 7. Examination of the potential splicing factors showed that curcumin specifically increased the protein and transcript levels of SRSF1. The increased SRSF1 protein was mainly nuclear and hyperphosphorylated. Interestingly, the curcumin effects on the SMN2 and SRSF1 transcripts were inhibited by a protein deacetylase inhibitor, trichostatin A. Moreover, in support of its role in the SMN2 splicing, knocking down SRSF1 reduced the inclusion of SMN2 exon 7. Thus, curcumin appears to have multiple effects on the SMN2 transcript and its splicing regulators, including the change of alternative splicing and transcript/protein level as well as phosphorylation. Protein deacetylases and phosphatases are likely involved in these effects. Interestingly, the effects all seem to favor production of the SMN2_E7+ transcript in SMA patient cells.

To identify the genetic etiology in a Chinese patient with neurofibromatosis type 1 (NF-1).All coding exons and the flanking sequences of neurofibromin 1 (NF1) gene from the patient were captured, individually barcoded and subjected to HiSeq2000 high-throughput sequencing. Suspected mutation was validated in the nuclear family members with Sanger sequencing.A novel indel mutation, c.789_790delAGinsT, was identified in the exon 8 of the NF1 gene in the patient but not in her asymptomatic parents. The mutation was predicted to have caused shifting of the reading frame and a premature downstream stop codon (p.K263Nfs*18). Two known polymorphisms, c.888+108 CT (rs2953000) and c.888+118 GT (rs2952999), was detected in the flanking of the indel mutation in the patient and her father. Sequencing chromatogram for the family indicates that above changes are located on the same chromosome.The c.789_790delAGinsT, as a de novo mutation occurring on the paternally derived chromosome, is most likely to be causative for the disease. Compared with Sanger sequencing, targeted next-generation sequencing is more efficient and can dramatically reduce the cost for the genetic testing of NF-1.

Succinic semialdehyde dehydrogenase (SSADH) deficiency is a rare autosomal recessive disorder that affects the degradation of γ-aminobutyric acid (GABA). Only a few cases of SSADH deficiency have been documented in mainland China and prenatal diagnosis has not been performed. SSADH deficiency in four patients (three girls and one boy) from four unrelated Chinese families was detected by selective screening at the age of 50days to 1year. Four patients were admitted due to intractable seizures and psychomotor retardation. Their urine 4-hydroxybutyric acid was significantly elevated. Seven mutations in their ALDH5A1 gene were identified, of which the following six were novel: c.127-128insGGCCC (p.Q43Rfs*50), [corrected] c.615delT (p.F206Sfs*5), c.1313T>C (p.L438P), c.1568C>T (p.S523F), 1383-2delA and a 0.15-Mb deletion harboring ALDH5A1. Only one mutation, c.820C>T, had been previously reported. Three mothers of Patients 1-3 underwent amniocentesis during their third pregnancy and the fetuses were not affected by SSADH deficiency. Normal development and urine organic acid levels of the infants confirmed the prenatal diagnosis after birth.

The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development., Author Summary In flowering plants, the proper transition from vegetative growth to flowering is critical for their reproductive success and must be controlled precisely. Multiple genes have been shown to regulate the floral transition in response to environmental and endogenous cues. Among them is SHORT VEGETATIVE PHASE (SVP), a key flowering repressor gene in Arabidopsis. SVP is highly expressed during the vegetative phase to promote growth, but the mechanism by which the high expression level of SVP is maintained remains unknown. Here, we report a genome-wide study to examine the functional interplay between the BRM chromatin remodeler and the PcG proteins that catalyze trimethylation of lysine 27 on histone H3 (H3K27me3), a histone mark normally associated with transcriptionally repressed genes. We identify BRM as a direct upstream activator of SVP. BRM acts to keep the levels of H3K27me3 low at the SVP locus by inhibiting the binding and activities of the PcG proteins. Thus, our work identifies a previously unknown mechanism in regulation of flowering time and demonstrates the power of genome-wide approaches in dissecting regulatory networks controlling plant development.

Isolated oral clefts, including cleft lip with/without cleft palate (CL/P) and cleft palate (CP), have a complex and heterogeneous etiology. Case-parent trios from three populations were used to study genes spanning chromosome 2, where single nucleotide polymorphic (SNP) markers were analyzed individually and as haplotypes. Case-parent trios from three populations (74 from Maryland, 64 from Singapore and 95 from Taiwan) were genotyped for 962 SNPs in 104 genes on chromosome 2, including two well-recognized candidate genes: TGFA and SATB2. Individual SNPs and haplotypes (in sliding windows of 2-5 SNPs) were used to test for linkage and disequilibrium separately in CL/P and CP trios. A novel candidate gene (ZNF533) showed consistent evidence of linkage and disequilibrium in all three populations for both CL/P and CP. SNPs in key regions of ZNF533 showed considerable variability in estimated genotypic odds ratios and their significance, suggesting allelic heterogeneity. Haplotype frequencies for regions of ZNF533 were estimated and used to partition genetic variance into among-and within-population components. Wright's fixation index, a measure of genetic diversity, showed little difference between Singapore and Taiwan compared with Maryland. The tensin-1 gene (TNS1) also showed evidence of linkage and disequilibrium among both CL/P and CP trios in all three populations, albeit at a lower level of significance. Additional genes (VAX2, GLI2, ZHFX1B on 2p; WNT6-WNT10A and COL4A3-COL4A4 on 2q) showed consistent evidence of linkage and disequilibrium only among CL/P trios in all three populations, and TGFA showed significant evidence in two of three populations.

AIM To report the clinical experience during collecting sperm samples in the Fragile X syndrome (FXS) male patients. METHODS Two different polymerase chain reaction (PCR) based methods were used for the molecular diagnosis of FXS. Sperm collection was done mostly according to the laboratory manual of the World Health Organization. RESULTS We failed to collect sperm samples from five Fragile X subjects aged 18-60 years as a result of an unexpected erectile dysfunction (ED). Multiple examinations of the same subject at different times, and of different subjects from different provinces examined by different physicians, showed the same result consistently in all the five subjects. CONCLUSION Erectile reflex is an instinctive response in all healthy males. The absence of erection can be caused by hormonal, physical or neuronal malfunction. As hormonal profiles were reported to be generally normal in Fragile X men, we propose that an unknown physical factor or the neuronal circuit, or both, underlying the erection is compromised. The finding of this symptom in Fragile X patients may help better understand the clinical spectrum and pathogeneses of the disease.

Seed storage proteins commonly comprise various groups of multiple isoforms encoded by different gene families. Arachin (11S globulin), conarachin (7S globulin) and conglutin (albumin) are the three major storage proteins in peanut (Arachis hypogaea L.). In order to isolate peanut seed storage protein genes, we sequenced and analyzed more than 400 randomly selected clones from a cDNA library derived from mid-maturation stage cotyledons of peanut. Our analysis indicated that arachin isoform precursors were encoded by at least five genes, whereas conglutins were encoded by more than six genes. The full-length cDNAs that encode isoforms of five arachin and six conglutin precursors were obtained. Among these cDNA clones, the deduced polypeptide of Ara 1 has about 99% similarity to that of peanut allergen Ara h 3. In addition, the deduced amino acid sequences of three conglutins, AhCONG 1, AhCONG 2 and AhCONG 5, are identical to those of peanut allergens Ara h 2.02, Ara h 2 and Ara h 6, respectively. Our study identified four new members (Ara 2 to Ara 5) of arachin family and two new members (AhCONG 3 and AhCONG 6) of conglutin family in peanut.