FGFR1 SUMOylation coordinates endothelial angiogenic signaling in angiogenesis.

Angiogenesis contributes fundamentally to embryonic development, tissue homeostasis, and wound healing. Basic fibroblast growth factor (FGF2) is recognized as the first proangiogenic molecule discovered, and it facilitates angiogenesis by activating FGF receptor 1 (FGFR1) signaling in endothelial cells. However, the precise roles of FGFR and the FGF/FGFR signaling axis in angiogenesis remain unclear, especially because of the contradictory phenotypes of in vivo FGF and FGFR gene deficiency models. Our previous study results suggested a potential role of posttranslational small ubiquitin-like modifier modification (SUMOylation), with highly dynamic regulatory features, in vascular development and disorder. Here, we identified SENP1-regulated endothelial FGFR1 SUMOylation at conserved lysines responding to proangiogenic stimuli, while SENP1 functioned as the deSUMOylase. Hypoxia-enhanced FGFR1 SUMOylation restricted the tyrosine kinase activation of FGFR1 by modulating the dimerization of FGFR1 and FGFR1 binding with its phosphatase PTPRG. Consequently, it facilitated the recruitment of FRS2α to VEGFR2 but limited additional recruitment of FRS2α to FGFR1, supporting the activation of VEGFA/VEGFR2 signaling in endothelial cells. Furthermore, SUMOylation-defective mutation of FGFR1 resulted in exaggerated FGF2/FGFR1 signaling but suppressed VEGFA/VEGFR2 signaling and the angiogenic capabilities of endothelial cells, which were rescued by FRS2α overexpression. Reduced angiogenesis and endothelial sprouting in mice bearing an endothelial-specific, FGFR1 SUMOylation-defective mutant confirmed the functional significance of endothelial FGFR1 SUMOylation in vivo. Our findings identify the reversible SUMOylation of FGFR1 as an intrinsic fine-tuned mechanism in coordinating endothelial angiogenic signaling during neovascularization; SENP1-regulated FGFR1 SUMOylation and deSUMOylation controls the competitive recruitment of FRS2α by FGFR1 and VEGFR2 to switch receptor-complex formation responding to hypoxia and normoxia angiogenic environments.

Construction of a High-Density Paulownia Genetic Map and QTL Mapping of Important Phenotypic Traits Based on Genome Assembly and Whole-Genome Resequencing.

Quantitative trait locus (QTL) mapping based on a genetic map is a very effective method of marker-assisted selection in breeding, and whole-genome resequencing is one of the useful methods to obtain high-density genetic maps. In this study, the hybrid assembly of Illumina, PacBio, and chromatin interaction mapping data was used to construct high-quality chromosomal genome sequences of Paulownia fortunei, with a size of 476.82 Mb, a heterozygosity of 0.52%, and a contig and scaffold N50s of 7.81 Mb and 21.81 Mb, respectively. Twenty scaffolds with a total length of 437.72 Mb were assembled into 20 pseudochromosomes. Repeat sequences with a total length of 243.96 Mb accounted for 51.16% of the entire genome. In all, 26,903 protein-coding gene loci were identified, and 26,008 (96.67%) genes had conserved functional motifs. Further comparative genomics analysis preliminarily showed that the split of P. fortunei with Tectona grandis likely occurred 38.8 (33.3-45.1) million years ago. Whole-genome resequencing was used to construct a merged genetic map of 20 linkage groups, with 2993 bin markers (3,312,780 SNPs), a total length of 1675.14 cm, and an average marker interval of 0.56 cm. In total, 73 QTLs for important phenotypic traits were identified (19 major QTLs with phenotypic variation explained ≥ 10%), including 10 for the diameter at breast height, 7 for the main trunk height, and 56 for branch-related traits. These results not only enrich P. fortunei genomic data but also form a solid foundation for fine QTL mapping and key marker/gene mining of Paulownia, which is of great significance for the directed genetic improvement of these species.

Association of chrononutrition patterns with biological aging: evidence from a nationally representative cross-sectional study.

Previous studies mostly focused on the benefits of caloric restriction and fasting on longevity. However, whether the timing and frequency of eating affect aging remains unclear. Here, we investigated the associations between chrononutrition patterns and biological aging, and explored whether and to what extent dietary inflammation mediated this association. 16 531 adults aged 20 to 84 years from the National Health and Nutrition Examination Survey were collected. Chrononutrition patterns were determined with two 24-hour dietary recalls. Phenotypic age was calculated to reflect the biological aging status. The dietary inflammatory index (DII) was used to assess the dietary inflammation. After adjustment of the survey weight and multiple covariates including total energy intake, participants in the third tertile of the time of the first meal (mean 10 : 26) exhibited more advanced biological age (ß 0.64; 95% CI, 0.26-1.00) and a higher incidence of accelerated aging (odds ratio (OR) 1.25; 95% CI, 1.06-1.47) compared to those of the first tertile (mean 6 : 14). Higher eating frequency was associated with delayed biological aging in both multivariable linear (ß -0.31; 95% CI, -0.44 to -0.19) and logistic regression model (OR 0.90; 95% CI, 0.85-0.95). Furthermore, we found that DII rather than metabolic factors mediated the inverse association between eating frequency and biological aging (mediation proportion 24.67%; 95% CI, 19.83%-32.00%). Our findings demonstrated the association between chrononutrition patterns and biological aging among the US general population and the potential role of dietary inflammation in this association, suggesting that modifying chrononutrition patterns may be a practical and cost-effective strategy for combating aging.

Circ_0007386 Promotes the Progression of Hepatocellular Carcinoma Through the miR-507/ CCNT2 Axis.

Background: Circular RNAs (circRNAs) have been shown to play a crucial role in the initiation and development of Hepatocellular carcinoma (HCC). However, the mechanism and function of circ_0007386 in HCC are still unknown. Methods: Circ_0007386 expression level in HCC tissues, and HCC cell lines was further analyzed by qRT-PCR. Agarose gel electrophoresis and Sanger sequencing were used to figure out the structure of circ_0007386. The involvement of circ_0007386 in HCC development was evaluated by experimental investigations conducted in both laboratory settings (in vitro) and living organisms (in vivo). RNA immunoprecipitation, Western blotting, luciferase reporter assay and fluorescence in situ hybridization (FISH) were applied for finding out the interaction among circ_0007386, miR-507 and CCNT2. To assess the connection between circ_0007386 and lenvatinib resistance, lenvatinib-resistant HCC cell lines were employed. Results: The expression of circ_0007386 was found to increase in HCC tissues, and it was observed to be associated with a worse prognosis. Overexpression of circ_0007386 stimulated HCC cells proliferation, invasion, migration and the epithelial-mesenchymal transition (EMT) while silencing of circ_0007386 resulted in the opposite effect. Mechanistic investigations revealed that circ_0007386 acted as a competing endogenous RNA of miR-507 to prevent CCNT2 downregulation. Downregulating miR-507 or overexpressing CCNT2 could reverse phenotypic alterations that originated from inhibiting of circ_0007386. Importantly, circ_0007386 determines the resistance of hepatoma cells to lenvatinib treatment. Conclusion: Circ_0007386 advanced HCC progression and lenvatinib resistance through the miR-507/ CCNT2 axis. Meanwhile, circ_0007386 served as a potential biomarker and therapeutic target in HCC patients.

Pooled screening with next-generation gene editing tools.

Pooled screening creates a pool of cells with genetic variants, allowing for the simultaneous examination for changes in behavior or function. By selectively inducing mutations or perturbing expression, it enables scientists to systematically investigate the function of genes or genetic elements. Emerging gene editing tools, such as CRISPR, coupled with advances in sequencing and computational capabilities, provide growing opportunities to understand biological processes in humans, animals, and plants as well as to identify potential targets for therapeutic interventions and agricultural research. In this review, we highlight the recent advances of pooled screens using next-generation gene editing tools along with relevant challenges and describe potential future directions of this technology.

Function and Cryo-EM structures of broadly potent bispecific antibodies against multiple SARS-CoV-2 Omicron sublineages.

The SARS-CoV-2 variant, Omicron (B.1.1.529), rapidly swept the world since its emergence. Compared with previous variants, Omicron has a high number of mutations, especially those in its spike glycoprotein that drastically dampen or abolish the efficacy of currently available vaccines and therapeutic antibodies. Several major sublineages of Omicron evolved, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75, which rapidly changing the global and regional landscape of the pandemic. Although vaccines are available, therapeutic antibodies remain critical for infected and especially hospitalized patients. To address this, we have designed and generated a panel of human/humanized therapeutic bispecific antibodies against Omicron and its sub-lineage variants, with activity spectrum against other lineages. Among these, the top clone CoV2-0213 has broadly potent activities against multiple SARS-CoV-2 ancestral and Omicron lineages, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75. We have solved the cryo-EM structure of the lead bi-specific antibody CoV-0213 and its major Fab arm MB.02. Three-dimensional structural analysis shows distinct epitope of antibody - spike receptor binding domain (RBD) interactions and reveals that both Fab fragments of CoV2-0213 can simultaneously target one single spike RBD or two adjacent ones in the same spike trimer, further corroborating its mechanism of action. CoV2-0213 represents a unique and potent broad-spectrum SARS-CoV-2 neutralizing bispecific antibody (nbsAb) against the currently circulating major Omicron variants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5). CoV2-0213 is primarily human and ready for translational testing as a countermeasure against the ever-evolving pathogen.

Exosome-transported circHDAC1_004 Promotes Proliferation, Migration, and Angiogenesis of Hepatocellular Carcinoma by the miR-361-3p/NACC1 Axis.

Background and Aims: Hepatocellular carcinoma (HCC) is among the most common malignant tumors globally. Circular RNAs (circRNAs), as a type of noncoding RNAs, reportedly participate in various tumor biological processes. However, the role of circHDAC1_004 in HCC remains unclear. Thus, we aimed to explore the role and the underlying mechanisms of circHDAC1_004 in the development and progression of HCC. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect circHDAC1_004 expression (circ_0005339) in HCC. Sanger sequencing and agarose gel electrophoresis were used to determine the structure of circHDAC1_004. In vitro and in vivo experiments were used to determine the biological function of circHDAC1_004 in HCC. Herein, qRT-PCR, RNA immunoprecipitation, western blotting, and a luciferase reporter assay were used to explore the relationships among circHDAC1_004, miR-361-3p, and NACC1. Results: circHDAC1_004 was upregulated in HCC and significantly associated with poor overall survival. circHDAC1_004 promoted HCC cell proliferation, stemness, migration, and invasion. In addition, circHDAC1_004 upregulated human umbilical vein endothelial cells (HUVECs) and promoted angiogenesis through exosomes. circHDAC1_004 promoted NACC1 expression and stimulated the epithelial-mesenchymal transition pathway by sponging miR-361-3p. Conclusions: We found that circHDAC1_004 overexpression enhanced the proliferation, stemness, and metastasis of HCC via the miR-361-3p/NACC1 axis and promoted HCC angiogenesis through exosomes. Our findings may help develop a possible therapeutic strategy for HCC.

Vaginal Microbiota Changes in Patients With Premature Ovarian Insufficiency and Its Correlation With Ovarian Function.

Objectives: To reveal the characteristics of vaginal microbiota in premature ovarian insufficiency (POI) patients and their relationship with ovarian function. Materials and Methods: In this case-control study, the vaginal bacterial composition of 30 POI patients and 26 healthy women of comparable age was assessed by 16S rRNA gene sequencing targeting the V3-V4 hypervariable regions. The metabolic functions of vaginal microflora were preliminarily predicted through the PICRUSt2 analysis. Redundancy analysis and Spearman's correlation analyzed the relationships between vaginal microbiota and ovarian function indicators. Results: Actinobacteria, Atopobium, and Gardnerella were significantly increased in POI patients. Their increments were significantly negatively correlated with anti-müllerian hormone (AMH) and inhibin B, and positively correlated with follicle-stimulating hormone (FSH) and luteinizing hormone (LH). While Bifidobacterium was significantly decreased in POI patients. Its relative abundance was significantly positively correlated with AMH and negatively correlated with FSH and LH. Then, POI patients included in this study were divided into POI (25 < FSH ≤ 40) (n = 9) and premature ovarian failure (POF) (FSH > 40) (n = 21) subgroups according to serum FSH levels. Compared with the controls, Firmicutes and Lactobacillus were significantly decreased only in POF (FSH > 40) patients, while no difference was observed in POI (25 < FSH ≤ 40) patients. Lactobacillus was negatively correlated with FSH. Firmicutes was significantly reduced and Actinobacteria was significantly increased in POF (FSH > 40) patients compared with POI (25 < FSH ≤ 40) patients. The key bacterial taxa Gardnerella and Atopobium showed potency in predicting POI. Conclusions: Here we demonstrated significant changes in the vaginal microbiota of POI patients, and these changes were significantly correlated with reduced ovarian reserve, endocrine disruption, and symptoms of perimenopausal syndrome. Differences in vaginal microbiota between POI (25 < FSH ≤ 40) and POF (FSH > 40) patients were also identified. These findings may provide new evidence for the relationship between vaginal microbiota and ovarian function.

Current Animal Model Systems for Ovarian Aging Research.

Ovarian aging leads to menopause, loss of fertility and other disorders in multiple organs, which brings great distress to women. For ethical reasons, it is impossible to use humans as direct study subjects for aging research. Therefore, biomedical researchers have employed different non-human organisms to study ovarian aging, including worms, fruit flies, fishes, amphibians, birds, mice, rats, cavies, rabbits, pigs, sheep, cows, horses, monkeys, and apes. Because each of these model organisms has its own features, multiple factors, such as size, anatomical structure, cost, ease of operation, fertility, generation time, lifespan, and gene heredity, should be carefully considered when selecting a model system to study ovarian aging. An appropriate model organism would help researchers explore the risk factors and elucidate molecular mechanisms underlying declined ovarian functions, which might be conducive to preventing or delaying the ovarian aging process. This article will offer an overview on several currently available and commonly used model organisms for ovarian aging research by comparing their pros and cons. In doing so, we hope to provide useful information for ovarian aging researchers.

Recent Advances in Second Near-Infrared Region (NIR-II) Fluorophores and Biomedical Applications.

Fluorescence imaging technique, characterized by high sensitivity, non-invasiveness and no radiation hazard, has been widely applicated in the biomedical field. However, the depth of tissue penetration is limited in the traditional (400-700 nm) and NIR-I (the first near-infrared region, 700-900 nm) imaging, which urges researchers to explore novel bioimaging modalities with high imaging performance. Prominent progress in the second near-infrared region (NIR-II, 1000-1700 nm) has greatly promoted the development of biomedical imaging. The NIR-II fluorescence imaging significantly overcomes the strong tissue absorption, auto-fluorescence as well as photon scattering, and has deep tissue penetration, micron-level spatial resolution, and high signal-to-background ratio. NIR-II bioimaging has been regarded as the most promising in vivo fluorescence imaging technology. High brightness and biocompatible fluorescent probes are crucial important for NIR-II in vivo imaging. Herein, we focus on the recently developed NIR-II fluorescent cores and their applications in the field of biomedicine, especially in tumor delineation and image-guided surgery, vascular imaging, NIR-II-based photothermal therapy and photodynamic therapy, drug delivery. Besides, the challenges and potential future developments of NIR-II fluorescence imaging are further discussed. It is expected that our review will lay a foundation for clinical translation of NIR-II biological imaging, and inspire new ideas and more researches in this field.

Full-length SMRT transcriptome sequencing and microsatellite characterization in Paulownia catalpifolia.

Paulownia catalpifolia is an important, fast-growing timber species known for its high density, color and texture. However, few transcriptomic and genetic studies have been conducted in P. catalpifolia. In this study, single-molecule real-time sequencing technology was applied to obtain the full-length transcriptome of P. catalpifolia leaves treated with varying degrees of drought stress. The sequencing data were then used to search for microsatellites, or simple sequence repeats (SSRs). A total of 28.83 Gb data were generated, 25,969 high-quality (HQ) transcripts with an average length of 1624 bp were acquired after removing the redundant reads, and 25,602 HQ transcripts (98.59%) were annotated using public databases. Among the HQ transcripts, 16,722 intact coding sequences, 149 long non-coding RNAs and 179 alternative splicing events were predicted, respectively. A total of 7367 SSR loci were distributed throughout 6293 HQ transcripts, of which 763 complex SSRs and 6604 complete SSRs. The SSR appearance frequency was 28.37%, and the average distribution distance was 5.59 kb. Among the 6604 complete SSR loci, 1-3 nucleotide repeats were dominant, occupying 97.85% of the total SSR loci, of which mono-, di- and tri-nucleotide repeats were 44.68%, 33.86% and 19.31%, respectively. We detected 112 repeat motifs, of which A/T (42.64%), AG/CT (12.22%), GA/TC (9.63%), GAA/TTC (1.57%) and CCA/TGG (1.54%) were most common in mono-, di- and tri-nucleotide repeats, respectively. The length of the repeat SSR motifs was 10-88 bp, and 4997 (75.67%) were ≤ 20 bp. This study provides a novel full-length transcriptome reference for P. catalpifolia and will facilitate the identification of germplasm resources and breeding of new drought-resistant P. catalpifolia varieties.

Reproductive risk factors for angiographic obstructive coronary artery disease among postmenopausal women.

OBJECTIVE: Reproductive factors are female-specific coronary artery disease (CAD) risk factors. However, the importance of reproductive factors in angiographic obstructive CAD in postmenopausal women remains uncertain. This study aimed to compare reproductive factors between postmenopausal women with no apparent CAD, nonobstructive CAD, and obstructive CAD and identify reproductive risk factors for obstructive CAD. METHODS: In this hospital-based cross-sectional study, 1,474 postmenopausal women, admitted with chest pain and referred for invasive coronary angiography were enrolled between April 2013 and October 2018. RESULTS: Adjusted odds ratio (95% CI) for obstructive CAD were 1.81 (1.03-3.17) for multigravidity (three or more pregnancies), 1.77 (1.14-2.76) for early menopause (≤40 y old), and 1.72 (1.26-2.35) for short reproductive life span (≤30 y). Each additional year in age at menopause or reproductive life span was associated with a 4% reduction in obstructive CAD risk in postmenopausal women (odds ratio, 0.96; 95% CI, 0.94-0.99; P = 0.011). The other reproductive factors, including parity, age at first birth, spontaneous abortion, induced abortion, stillbirth, hypertensive disorders of pregnancy, gestational diabetes mellitus, and age at menarche, were not correlated with obstructive CAD risk in postmenopausal women. CONCLUSIONS: Multigravidity (three or more pregnancies), early menopause, and a shorter reproductive life span were independent risk factors of angiographic obstructive CAD among postmenopausal women, which suggested that pregnancy and ovarian function may be important for the early identification and prevention of increased risk of female angiographic obstructive CAD.

Distinct physiological and transcriptional responses of leaves of paper mulberry (Broussonetia kazinoki × B. papyrifera) under different nitrogen supply levels.

Paper mulberry, a vigorous pioneer species used for ecological reclamation and a high-protein forage plant for economic development, has been widely planted in China. To further develop its potential value, it is necessary to explore the regulatory mechanism of nitrogen metabolism for rational nitrogen utilization. In this study, we investigated the morphology, physiology and transcriptome of a paper mulberry hybrid (Broussonetia kazinoki × B. papyrifera) in response to different nitrogen concentrations. Moderate nitrogen promoted plant growth and biomass accumulation. Photosynthetic characteristics, concentration of nitrogenous compounds and activities of enzymes were stimulated under nitrogen treatment. However, these enhancements were slightly or severely inhibited under excessive nitrogen supply. Nitrite reductase and glutamate synthase were more sensitive than nitrate reductase and glutamine synthetase and more likely to be inhibited under high nitrogen concentrations. Transcriptome analysis of the leaf transcriptome identified 161,961 unigenes. The differentially expressed genes associated with metabolism of nitrogen, alanine, aspartate, glutamate and glycerophospholipid showed high transcript abundances after nitrogen application, whereas those associated with glycerophospholipid, glycerolipid, amino sugar and nucleotide sugar metabolism were down-regulated. Combined with weighted gene coexpression network analysis, we uncovered 16 modules according to similarity in expression patterns. Asparagine synthetase and inorganic pyrophosphatase were considered two hub genes in two modules, which were associated with nitrogen metabolism and phosphorus metabolism, respectively. The expression characteristics of these genes may explain the regulation of morphological, physiological and other related metabolic strategies harmoniously. This multifaceted study provides valuable insights to further understand the mechanism of nitrogen metabolism and to guide utilization of paper mulberry.

Specific Small-Molecule NIR-II Fluorescence Imaging of Osteosarcoma and Lung Metastasis.

Osteosarcoma is an aggressive tumor of mesenchymal origin that is more likely to spread to the lung than others, with a major impact on patients' prognosis. The optimal imaging method that can reliably detect or exclude pulmonary metastases from osteosarcoma is still scarce. Herein, two homologous types of fluorescent probes CH1055-PEG-PT and CH1055-PEG-Affibody, which show highly promising results for targeting imaging of osteosarcoma and its lung metastasis, respectively, are designed and synthesized. It is found that the NIR-II imaging quality of CH1055-PEG-PT is far superior to that of computed tomography for the early in vivo 143B tumor imaging, and this probe-guided surgery for accurate resection of 143B tumor is further performed. The high-resolution visualization of primary and micrometastatic lung lesions of osteosarcoma by using CH1055-PEG-Affibody is also demonstrated. Therefore, the attractive imaging properties of CH1055-PEG-PT and CH1055-PEG-Affibody, including high levels of uptakes, and high spatial and temporal resolution, open up opportunities for molecular imaging and clinical translation of osteosarcoma and its lung metastasis in the unique second near-infrared window.

Ectopic expression of EuWRI1, encoding a transcription factor in E. ulmoides, changes the seeds oil content in transgenic tobacco.

The WRINKLED1 (WRI1) gene is a well-established key transcriptional regulator involved in the regulation of fatty acid biosynthesis in developing seeds. In this study, a new WRI1 gene was isolated from seeds of Eucommia ulmoides and named EuWRI1. A close link between gibberellins signaling and EuWRI1 gene expression was suggested in this study. Functional characterization of EuWRI1 was elucidated through seed-specific expression in tobacco. In transgenic tobacco, the expression of EuWRI1 in eight independent transgenic lines was detected by semiquantitative RT-PCR. The relative mRNA accumulation of genes encoding enzymes involved in fatty acid biosynthesis (biotin carboxyl carrier protein and keto-ACP synthase 1) was also assayed in tobacco seeds. Analysis of the seeds oil content and starch content indicated that the transgenic lines showed a significant increase in seeds oil content, whereas starch content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (16:0), linoleic acid (18:2) and linolenic acid (18:3) increased significantly in seeds of transgenic tobacco lines, but stearic acid (18:0) levels significantly declined. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:337-346, 2018.

Characterization of Glycolytic Pathway Genes Using RNA-Seq in Developing Kernels of Eucommia ulmoides.

Eucommia ulmoides Oliver, the only member of the Eucommiaceae family, is a rare and valuable tree used to produce a highly valued traditional Chinese medicine and contains α-linolenic acid (ALA) up to 60% of the total fatty acids in the kernels (embryos). Glycolysis provides both cellular energy and the intermediates for other biosynthetic processes. However, nothing was known about the molecular basis of the glycolytic pathway in E. ulmoides kernels. The purposes of this study were to identify novel genes of E. ulmoides related to glycolytic metabolism and to analyze the expression patterns of selected genes in the kernels. Transcriptome sequencing based on the Illumina platform generated 96,469 unigenes in four cDNA libraries constructed using RNAs from 70 and 160 days after flowering kernels of both low- and high-ALA varieties. We identified and characterized the digital expression of 120 unigenes coding for 24 protein families involved in kernel glycolytic pathway. The expression levels of glycolytic genes were generally higher in younger kernels than in more mature kernels. Importantly, several unigenes from kernels of the high-ALA variety were expressed more than those from the low-ALA variety. The expression of 10 unigenes encoding key enzymes in the glycolytic pathway was validated by qPCR using RNAs from six kernel stages of each variety. The qPCR data were well consistent with their digital expression in transcriptomic analyses. This study identified a comprehensive set of genes for glycolytic metabolism and suggests that several glycolytic genes may play key roles in ALA accumulation in the kernels of E. ulmoides.

Transcriptomic identification and expression of starch and sucrose metabolism genes in the seeds of Chinese chestnut (Castanea mollissima).

The Chinese chestnut (Castanea mollissima) seed provides a rich source of carbohydrates as food and feed. However, little is known about starch biosynthesis in the seeds. The objectives of this study were to determine seed composition profiles and identify genes involved in starch and sucrose metabolism. Metabolite analysis showed that starch was the major component and rapidly accumulated during seed endosperm development. Amylopectin was approximately 3-fold of amylose content in chestnut starch. Illumina platform-based transcriptome sequencing generated 56671 unigenes in two cDNA libraries from seed endosperms collected at 45 and 75 days after flowering (DAF). A total of 1537 unigenes showed expression differences ≥2-fold in the two stages of seeds including 570 up-regulated and 967 down-regulated unigenes. One hundred and fifty-two unigenes were identified as involved in starch and sucrose metabolism, including 1 for glycogenin glucosyltransferase, 4 for adenylate transporter (brittle1-type), 3 for ADP-glucose pyrophosphorylase (AGP, not brittle2- or shrunken2-type), 3 for starch synthase (SS), 2 for starch branching enzyme, 5 for starch debranching enzyme, 11 for sucrose synthase, and 3 for sucrose-phosphate synthase. Among them, 58 unigenes showed a ≥2-fold expression difference between the 45 and 75 DAF seeds including 11 up- and 47 down-regulated unigenes. The expression of 21 unigenes putatively coding for major enzymes in starch and sucrose metabolism was validated by qPCR using RNA from five seed stages. Expression profiles and correlation analysis indicated that the mRNA levels of AGP (large and small subunits), granule-bound SS2, and soluble SS1 and SS4 were well-correlated with starch accumulation in the seeds. This study suggests that the starch biosynthesis pathway in Chinese chestnut is similar to that of potato tuber/Arabidopsis leaf and differs from that of maize endosperm. The information provides valuable metabolite and genetic resources for future research in starch and sucrose metabolism in Chinese chestnut tree.