A haplotype-resolved genome assembly of Malus domestica 'Red Fuji'.

The 'Red Fuji' apple (Malus domestica), is one of the most important and popular economic crops worldwide in the fruit industry. Using PacBio HiFi long reads and Hi-C reads, we assembled a high-quality haplotype-resolved genome of 'Red Fuji', with sizes of 668.7 and 668.8 Mb, and N50 sizes of 34.1 and 31.4 Mb. About 97.2% of sequences were anchored in 34 chromosomes. We annotated both haploid genomes, identifying a total of 95,439 protein-coding genes in the two haplotype genomes, with 98% functional annotation. The haplotype-resolved genome of 'Red Fuji' apple stands as a precise benchmark for an array of analyses, such as comparative genomics, transcriptomics, and allelic expression studies. This comprehensive resource is paramount in unraveling variations in allelic expression, advancing quality improvements, and refining breeding efforts.

Chromosomal level genome assemblies of two Malus crabapple cultivars Flame and Royalty.

Malus hybrid 'Flame' and Malus hybrid 'Royalty' are representative ornamental crabapples, rich in flavonoids and serving as the preferred materials for studying the coloration mechanism. We generated two sets of high-quality chromosome-level and haplotype-resolved genome of 'Flame' with sizes of 688.2 Mb and 675.7 Mb, and those of 'Royalty' with sizes of 674.1 Mb and 663.6 Mb, all anchored to 17 chromosomes and with a high BUSCO completeness score nearly 99.0%. A total of 47,833 and 47,307 protein-coding genes were annotated in the two haplotype genomes of 'Flame', and the numbers of 'Royalty' were 46,305 and 46,920 individually. The assembled high-quality genomes offer new resources for studying the origin and adaptive evolution of crabapples and the molecular basis of the accumulation of flavonoids and anthocyanins, facilitating molecular breeding of Malus plants.

Two novel heterozygous truncating variants in NR4A2 identified in patients with neurodevelopmental disorder and brief literature review.

Pathogenic variants in the nuclear receptor superfamily 4 group A member 2 (NR4A2) cause an autosomal dominant neurodevelopmental disorder with or without seizures. Here, we described two patients presenting with developmental delay, language impairment, and attention-deficit hyperactivity disorder. Trio-based whole exome sequencing revealed two novel heterozygous variants, c.1541-2A > C and c.915C > A, in NR4A2. Both variants were identified as de novo and confirmed by Sanger sequencing. In vitro functional analyses were performed to assess their effects on expression of mRNA or protein. The canonical splicing variant c.1541-2A > C caused aberrant splicing, leading to the retention of intron 7 and a truncated protein due to an early termination codon within intron 7 with decreased protein expression, while the variant c.915C > A was shown to result in a shorter protein with increased expression level unexpectedly. The clinical and genetic characteristics of the previously published patients were briefly reviewed for highlighting the potential link between mutations and phenotypes. Our research further confirms that NR4A2 is a disease-causing gene of neurodevelopmental disorders and suggests alterations in different domains of NR4A2 cause various severity of symptoms.

McMYB4 improves temperature adaptation by regulating phenylpropanoid metabolism and hormone signaling in apple.

Temperature changes affect apple development and production. Phenylpropanoid metabolism and hormone signaling play a crucial role in regulating apple growth and development in response to temperature changes. Here, we found that McMYB4 is induced by treatment at 28 °C and 18 °C, and McMYB4 overexpression results in flavonol and lignin accumulation in apple leaves. Yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs) further revealed that McMYB4 targets the promoters of the flavonol biosynthesis genes CHS and FLS and the lignin biosynthesis genes CAD and F5H. McMYB4 expression resulted in higher levels of flavonol and lignin biosynthesis in apple during growth at 28 °C and 18 °C than during growth at 23 °C. At 28 °C and 18 °C, McMYB4 also binds to the AUX/ARF and BRI/BIN promoters to activate gene expression, resulting in acceleration of the auxin and brassinolide signaling pathways. Taken together, our results demonstrate that McMYB4 promotes flavonol biosynthesis and brassinolide signaling, which decreases ROS contents to improve plant resistance and promotes lignin biosynthesis and auxin signaling to regulate plant growth. This study suggests that McMYB4 participates in the abiotic resistance and growth of apple in response to temperature changes by regulating phenylpropanoid metabolism and hormone signaling.

Animal wastes as fertilizers enhance growth of young walnut trees under soil drought conditions.

BACKGROUND: Using nutrient-rich animal wastes as organic fertilizers in agricultural practices is a sustainable method for soil amendment and avoiding environmental pollution. In order to evaluate their practical effect, we applied different proportions of animal waste as fertilizers to wet or dry soils that were either planted or not planted with young walnut trees. RESULTS: The results showed that animal waste could increase soil C accumulation and carbon to nitrogen (C/N) ratio and reduce soil organic nitrogen and total nitrogen contents as well as the nitrogen to phosphorus (N/P) ratio in the planted group soil. This framework of soil C and N composition (a high C/N ratio) resulted in high N and Mg contents as well as high Cu and Zn contents in the leaves of the young trees as well as a high dry matter weight/leaf N ratio, causing increased leaf photosynthesis, reduced transpiration and relatively high water use efficiency under soil drought conditions. Also, animal wastes as fertilizers caused the branching of walnut to switch from elongation growth to thickening growth under soil drought conditions. CONCLUSIONS: Principal component analysis and redundancy analysis demonstrated the mechanism by which the soil C/N ratio mediates the flux of available nutrients from the soil to the plant and thereby regulates plant dry matter accumulation and branching architecture under soil drought conditions. The results of this study provide new insights into the improvement of hilly soils using animal waste. © 2020 Society of Chemical Industry.

MiR399d and epigenetic modification comodulate anthocyanin accumulation in Malus leaves suffering from phosphorus deficiency.

Inorganic phosphorus (Pi) deficiency induces anthocyanin accumulation in the leaves of some plant species; however, the molecular mechanisms underlying this phenomenon have not been well characterized. Here, we showed that microRNA399d (miR399d), high-affinity Pi transporter McPHT1;4, and McMYB10 are strongly induced in Malus leaves suffering from Pi deficiency. By culturing explants of transiently transformed plants in MS medium under conditions of Pi sufficiency and Pi deficiency, miR399d and McPHT1;4 were shown to play essential roles in the response to Pi deficiency and to play positive roles in the regulation of anthocyanin biosynthesis. Silencing of McHDA6 expression and treatment with the inhibitor trichostatin A suggested that the low expression of McHDA6 simultaneously reduced the transcription of McMET1 and decreased the methylation level of the McMYB10 promoter; however, the expression of McMYB10 and anthocyanin content were increased. Bimolecular fluorescence complementation and yeast two-hybrid assays revealed that McHDA6 binds directly to McMET1 through its BAH2 and DNMT1-RFD domains. Based on the results of our study, we propose a mechanism for the molecular regulation of anthocyanin biosynthesis, namely, the miR399d and epigenetic modification comodulation model, to explain the phenomenon in which leaves turn red under conditions of Pi deficiency.

Ectopic Dpp signaling promotes stem cell competition through EGFR signaling in the Drosophila testis.

Stem cell competition could select the fittest stem cells and potentially control tumorigenesis. However, little is known about the underlying molecular mechanisms. Here, we find that ectopic Decapentaplegic (Dpp) signal activation by expressing a constitutively active form of Thickveins (TkvCA) in cyst stem cells (CySCs) leads to competition between CySCs and germline stem cells (GSCs) for niche occupancy and GSC loss. GSCs are displaced from the niche and undergo differentiation. Interestingly, we find that induction of TkvCA results in elevated expression of vein, which further activates Epidermal Growth Factor Receptor (EGFR) signaling in CySCs to promote their proliferation and compete GSCs out of the niche. Our findings elucidate the important role of Dpp signaling in regulating stem cell competition and tumorigenesis, which could be shed light on tumorigenesis and cancer treatment in mammals.

CHRNE compound heterozygous mutations in congenital myasthenic syndrome: A case report.

RATIONALE: Congenital myasthenic syndrome (CMSs) are a group of rare genetic disorders of the neurological junction, which can result in structural or functional weakness. Here, we characterized a case of CMS in order to clarify the diagnosis and expand the understanding of it. The molecular diagnosis had implications for choice of treatment and genetic counseling. PATIENT CONCERNS: A 3-year-old male patient with CMS had ptosis and limb weakness for 2 months after birth. Clinical course and electrophysiological, imaging, and genetic findings were assessed. Protein structure/function was predicted. A novel mutation of c.295C>T (exon 4) and another known mutation of c.442T>A (exon 5) were found in CHRNE. Both mutations localized in conserved sequences. The c.442T>A (p.C148S) missense mutation in CHRNE was predicted to be damaging/deleterious. The iterative threading assembly refinement (I-TASSER) server generated vastly different 3-dimensional (3D) atomic models based on protein sequences from wide-type and novel nonsense mutation of c.295C>T (p.R99X) in CHRNE. DIAGNOSES: The diagnosis of CMS with CHRNE mutations in Han Chinese was confirmed. INTERVENTIONS: The patient was given prednisone (10 mg, once daily, taken orally) and pyridostigmine (15 mg, three times a day, taken orally). OUTCOMES: The patient had a moderate response to prednisone and pyridostigmine. LESSONS: We expanded the genotype and phenotype of CMS with CHRNE mutations in Han Chinese and provided new insights into the molecular mechanism of CMS and help to the diagnosis and treatment of CMS.

Systematic Chemical Analysis Approach Reveals Superior Antioxidant Capacity via the Synergistic Effect of Flavonoid Compounds in Red Vegetative Tissues.

The flavonoid system comprises an abundance of compounds with multiple functions; however, their potential synergism in antioxidant function remains unclear. We established an approach using ever-red (RL) and ever-green leaves (GL) of crabapple cultivars during their development to determine interrelationships among flavonoid compounds. RL scored significantly better than GL in terms of the type, composition, and diversity of flavonoids than GL. Principal component analysis predicted flavonoids in RL to have positive interaction effects, and the total antioxidant capacity was significantly higher than the sum of antioxidant capacities of the individual compounds. This synergy was verified by the high antioxidant capacity in rat serum after feeding on red leaves. Our findings suggest that the synergistic effect is a result of the high transcription levels regulated by McMYBs in RL. In summary, individual flavonoids cooperate in a flavonoid system, thus producing a synergistic antioxidant effect, and the approach used herein can provide insights into the roles of flavonoids and other compounds in future studies.

Dopa-Responsive Dystonia in Han Chinese Patients: One Novel Heterozygous Mutation in GTP Cyclohydrolase 1 (GCH1) and Three Known Mutations in TH.

BACKGROUND This study aimed to clarify the diagnosis and expand the understanding of dopa-responsive dystonia (DRD). MATERIAL AND METHODS Relevant data from clinical diagnoses and genetic mutational analyses in 3 Han Chinese patients with sporadic DRD were collected and analyzed. Protein structure/function was predicted. RESULTS One novel mutation of c.679A>G (p.T227A) in GCH1 and 3 known mutations of c.457C>T (p.R153X), c.739G>A (p.G247S), and c.698G>A (p.R227H) in tyrosine hydroxylase (TH) have been found and predicted to be damaging or deleterious. All of the mutations were localized in conserved sequences. The iterative threading assembly refinement (I-TASSER) server generated three-dimensional (3D) atomic models based on protein sequences from the novel nonsense mutation of c.679A>G (p.T227A) in GCH1, which showed that residue 227 was located in the GCH1 active site. CONCLUSIONS Patients carrying different non-synonymous variants had remarkable variation in clinical phenotype. This study expands the spectrum of genotypes and phenotypes of DRD in the Han Chinese ethnicity, provides new insights into the molecular mechanism of DRD, and helps the diagnosis and treatment of DRD.

Flavonoid Accumulation Plays an Important Role in the Rust Resistance of Malus Plant Leaves.

Cedar-apple rust (Gymnosporangium yamadai Miyabe) is a fungal disease that causes substantial injury to apple trees and results in fruit with reduced size and quality and a lower commercial value. The molecular mechanisms underlying the primary and secondary metabolic effects of rust spots on the leaves of Malus apple cultivars are poorly understood. Using HPLC, we found that the contents of flavonoid compounds, especially anthocyanin and catechin, were significantly increased in rust-infected symptomatic tissue (RIT). The expression levels of structural genes and MYB transcription factors related to flavonoid biosynthesis were one- to seven-fold higher in the RIT. Among these genes, CHS, DFR, ANS, FLS and MYB10 showed more than a 10-fold increase, suggesting that these genes were expressed at significantly higher levels in the RIT. Hormone concentration assays showed that the levels of abscisic acid (ABA), ethylene (ETH), jasmonate (JA) and salicylic acid (SA) were higher in the RIT and were consistent with the expression levels of McNCED, McACS, McLOX and McNPR1, respectively. Our study explored the complicated crosstalk of the signal transduction pathways of ABA, ETH, JA and SA; the primary metabolism of glucose, sucrose, fructose and sorbitol; and the secondary metabolism of flavonoids involved in the rust resistance of Malus crabapple leaves.

MYBs affect the variation in the ratio of anthocyanin and flavanol in fruit peel and flesh in response to shade.

Fruit pigment accumulation, which represents an important indicator of nutrient quality and appearance value, is often affected by low light under rain, cloud, fog and haze conditions during the veraison period. It is not known whether continuous low light interferes with the production and accumulation of secondary metabolites in veraison fruit. In this paper, we measured pigments and the transcriptional level of genes related to secondary metabolites, i.e., flavonoid biosynthesis in the peel and flesh of Malus crabapple 'Radiant' fruit in response to normal light and shade from 10th July to 30th August. The results showed crosstalk between the flavonoid biosynthetic genes and the involvement of key transcription factors such as McMYB4, McMYB7, McMYB10, and McMYB16 in the regulation of the ratio of anthocyanins and flavanols, which accounted for the different colouration of the fruit peel and flesh under shade conditions. A model is proposed for the regulation of the flavonoid pathway in the peel and flesh of 'Radiant' fruit based on our study results. Moreover, the molecular mechanism for 'Radiant' fruit colouration provides reference information for understanding the light regulatory mechanism involved in the biosynthesis of flavonoids and for designing the next generation of apple breeding.

Mitochondrial DNA Rearrangement Spectrum in Brain Tissue of Alzheimer's Disease: Analysis of 13 Cases.

BACKGROUND: Mitochondrial dysfunction may play a central role in the pathologic process of Alzheimer's disease (AD), but there is still a scarcity of data that directly links the pathology of AD with the alteration of mitochondrial DNA. This study aimed to provide a comprehensive assessment of mtDNA rearrangement events in AD brain tissue. PATIENTS AND METHODS: Postmortem frozen human brain cerebral cortex samples were obtained from the Banner Sun Health Research Institute Brain and Body Donation Program, Sun City, AZ. Mitochondria were isolated and direct sequence by using MiSeq®, and analyzed by relative software. RESULTS: Three types of mitochondrial DNA (mtDNA) rearrangements have been seen in post mortem human brain tissue from patients with AD and age matched control. These observed rearrangements include a deletion, F-type rearrangement, and R-type rearrangement. We detected a high level of mtDNA rearrangement in brain tissue from cognitively normal subjects, as well as the patients with Alzheimer's disease (AD). The rate of rearrangements was calculated by dividing the number of positive rearrangements by the coverage depth. The rearrangement rate was significantly higher in AD brain tissue than in control brain tissue (17.9%versus 6.7%; p = 0.0052). Of specific types of rearrangement, deletions were markedly increased in AD (9.2% versus 2.3%; p = 0.0005). CONCLUSIONS: Our data showed that failure of mitochondrial DNA in AD brain might be important etiology of AD pathology.

Notch signaling downstream target E(spl)mbeta is dispensable for adult midgut homeostasis in Drosophila.

Adult tissue homeostasis is maintained by residential stem cells through the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs), and Notch signaling plays critical roles in the proliferation and differentiation of these ISCs. However, how Notch signaling downstream targets regulate ISC proliferation and differentiation still remains unclear. Here we find that Notch signaling downstream targets E(spl)mbeta and E(spl)malpha are differentially expressed in ISCs and their progeny. Interestingly, we find that midgut homeostasis is not affected in E(spl)mbeta null mutant. No obvious defects are observed in the intestines ectopically expressing E(spl)mbeta or E(spl)malpha. Importantly, we find that the defects in ISC proliferation and differentiation observed in Notch mutant cannot be rescued by ectopic expression of E(spl)mbeta or E(spl)malpha. Together, these data indicate that the proliferation and differentiation of ISCs are not regulated by individual Notch downstream target, but by different downstream targets collectively.

Arabidopsis pentatricopeptide repeat protein SOAR1 plays a critical role in abscisic acid signalling.

A dominant suppressor of the ABAR overexpressor, soar1-1D, from CHLH/ABAR [coding for Mg-chelatase H subunit/putative abscisic acid (ABA) receptor (ABAR)] overexpression lines was screened to explore the mechanism of the ABAR-mediated ABA signalling. The SOAR1 gene encodes a pentatricopeptide repeat (PPR) protein which localizes to both the cytosol and nucleus. Down-regulation of SOAR1 strongly enhances, but up-regulation of SOAR1 almost completely impairs, ABA responses, revealing that SOAR1 is a critical, negative, regulator of ABA signalling. Further genetic evidence supports that SOAR1 functions downstream of ABAR and probably upstream of an ABA-responsive transcription factor ABI5. Changes in the SOAR1 expression alter expression of a subset of ABA-responsive genes including ABI5. These findings provide important information to elucidate further the functional mechanism of PPR proteins and the complicated ABA signalling network.

Arabidopsis co-chaperonin CPN20 antagonizes Mg-chelatase H subunit to derepress ABA-responsive WRKY40 transcription repressor.

Our previous study demonstrated that a chloroplast co-chaperonin 20 (CPN20), one of the interaction partners of the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR), negatively regulates ABA signaling at the same node with ABAR but upstream of WRKY40 transcription repressor in Arabidopsis thaliana. In the present experiment, we showed that ABA directly inhibits the ABAR-CPN20 interaction, and also represses expression of CPN20, which depends on ABAR. CPN20 inhibits ABAR-WRKY40 interaction by competitively binding to ABAR. ABAR downregulates, but CPN20 upregulates, WRKY40 expression. The cpn20-1 mutation induces downregulation of WRKY40, and suppresses the upregulated level of WRKY40 due to the cch mutation in the ABAR gene. ABA-induced repressive effect of the WRKY40 gene is strengthened by downregulation of CPN20 but reduced by upregulation of CPN20. Together with our previously reported genetic data, we provide evidence that CPN20 functions through antagonizing the ABAR-WRKY40 coupled pathway, and ABA relieves this pathway of repression by inhibiting the ABAR-CPN20 interaction to activate ABAR-WRKY40 interaction.

Light-harvesting chlorophyll a/b-binding proteins, positively involved in abscisic acid signalling, require a transcription repressor, WRKY40, to balance their function.

The light-harvesting chlorophyll a/b-binding (LHCB) proteins are the apoproteins of the light-harvesting complex of photosystem II. In the present study, we observed that downregulation of any of the six LHCB genes resulted in abscisic acid (ABA)-insensitive phenotypes in seed germination and post-germination growth, demonstrating that LHCB proteins are positively involved in these developmental processes in response to ABA. ABA was required for full expression of different LHCB members and physiologically high levels of ABA enhanced LHCB expression. The LHCB members were shown to be targets of an ABA-responsive WRKY-domain transcription factor, WRKY40, which represses LHCB expression to balance the positive function of the LHCBs in ABA signalling. These findings revealed that ABA is an inducer that fine-tunes LHCB expression at least partly through repressing the WRKY40 transcription repressor in stressful conditions in co-operation with light, which allows plants to adapt to environmental challenges.

COP1 mediates the coordination of root and shoot growth by light through modulation of PIN1- and PIN2-dependent auxin transport in Arabidopsis.

When a plant germinates in the soil, elongation of stem-like organs is enhanced whereas leaf and root growth is inhibited. How these differential growth responses are orchestrated by light and integrated at the organismal level to shape the plant remains to be elucidated. Here, we show that light signals through the master photomorphogenesis repressor COP1 to coordinate root and shoot growth in Arabidopsis. In the shoot, COP1 regulates shoot-to-root auxin transport by controlling the transcription of the auxin efflux carrier gene PIN-FORMED1 (PIN1), thus appropriately tuning shoot-derived auxin levels in the root. This in turn directly influences root elongation and adapts auxin transport and cell proliferation in the root apical meristem by modulating PIN1 and PIN2 intracellular distribution in the root in a COP1-dependent fashion, thus permitting a rapid and precise tuning of root growth to the light environment. Our data identify auxin as a long-distance signal in developmental adaptation to light and illustrate how spatially separated control mechanisms can converge on the same signaling system to coordinate development at the whole plant level.

Import oligomers induce positive feedback to promote peroxisome differentiation and control organelle abundance.

A fundamental question in cell biology is how cells control organelle composition and abundance. Woronin bodies are fungal peroxisomes centered on a crystalline core of the self-assembled HEX protein. Despite using the canonical peroxisome import machinery for biogenesis, Woronin bodies are scarce compared to the overall peroxisome population. Here, we show that HEX oligomers promote the differentiation of a subpopulation of peroxisomes, which become enlarged and highly active in matrix protein import. HEX physically associates with the essential matrix import peroxin, PEX26, and promotes its enrichment in the membrane of differentiated peroxisomes. In addition, a PEX26 mutant that disrupts differentiation produces increased numbers of aberrantly small Woronin bodies. Our data suggest a mechanism where HEX oligomers recruit a key component of the import machinery, which promotes the import of additional HEX. This type of positive feedback provides a basic mechanism for the production of an organelle subpopulation of distinct composition and abundance.