Transcriptome and photosynthetic analyses provide new insight into the molecular mechanisms underlying heat stress tolerance in Rhododendron × pulchrum Sweet.

Rhododendron species provide excellent ornamental use worldwide, yet heat stress (HS) is one of the major threats to their cultivation. However, the intricate mechanisms underlying the photochemical and transcriptional regulations associated with the heat stress response in Rhododendron remain relatively unexplored. In this study, the analyses of morphological characteristics and chlorophyll fluorescence (ChlF) kinetics showed that HS (40 °C/35 °C) had a notable impact on both the donor's and acceptor's sides of photosystem II (PSII), resulting in reduced PSII activity and electron transfer capacity. The gradual recovery of plants observed following a 5-day period of culture under normal conditions indicates the reversible nature of the HS impact on Rhododendron × pulchrum. Analysis of transcriptome data unveiled noteworthy trends: four genes associated with photosynthesis-antenna protein synthesis (LHCb1, LHCb2 and LHCb3) and the antioxidant system (glutamate-cysteine ligase) experienced significant down-regulation in the leaves of R. × pulchrum during HS. Conversely, aseorbate peroxidase and glutathione S-transferase TAU 8 demonstrated an up-regulated pattern. Furthermore, six down-regulated genes (phos-phoenolpyruvate carboxylase 4, sedoheptulose-bisphosphatase, ribose-5-phosphate isomerase 2, high cyclic electron flow 1, beta glucosidase 32 and starch synthase 2) and two up-regulated genes (beta glucosidase 2 and UDP-glucose pyrophosphorylase 2) implicated in photosynthetic carbon fixation and starch/sucrose metabolism were identified during the recovery process. To augment these insights, a weighted gene co-expression network analysis yielded a co-expression network, pinpointing the hub genes correlated with ChlF dynamics' variation trends. The cumulative results showed that HS inhibited the synthesis of photosynthesis-antenna proteins in R. × pulchrum leaves. This disruption subsequently led to diminished photochemical activities in both PSII and PSI, albeit with PSI exhibiting heightened thermostability. Depending on the regulation of the reactive oxygen species scavenging system and heat dissipation, photoprotection sustained the recoverability of R. × pulchrum to HS.

A population-based study of precision health assessments using multi-omics network-derived biological functional modules.

Recent technological advances in multi-omics and bioinformatics provide an opportunity to develop precision health assessments, which require big data and relevant bioinformatic methods. Here we collect multi-omics data from 4,277 individuals. We calculate the correlations between pairwise features from cross-sectional data and then generate 11 biological functional modules (BFMs) in males and 12 BFMs in females using a community detection algorithm. Using the features in the BFM associated with cardiometabolic health, carotid plaques can be predicted accurately in an independent dataset. We developed a model by comparing individual data with the health baseline in BFMs to assess health status (BFM-ash). Then we apply the model to chronic patients and modify the BFM-ash model to assess the effects of consuming grape seed extract as a dietary supplement. Finally, anomalous BFMs are identified for each subject. Our BFMs and BFM-ash model have huge prospects for application in precision health assessment.

ZeMYB9 regulates cyanidin synthesis by activating the expression of flavonoid 3'-hydroxylase gene in Zinnia elegans.

Petal color in Zinnia elegans is characterized mainly by anthocyanin accumulation. The difference in the content of anthocyanins, especially cyanidins, affects petal coloration in Z. elegans, but the underlying regulatory mechanism remains elusive. Here, we report one R2R3-MYB transcription factor from subgroup 6, ZeMYB9, acting as a positive regulator of anthocyanin accumulation in Z. elegans. Up-regulated expression of ZeMYB9 and flavonoid 3'-hydroxylase gene (ZeF3'H) was detected in the cultivar with higher cyanidin content. ZeMYB9 could specifically activate the promoter of ZeF3'H, and over-expression of ZeMYB9 induces much greater anthocyanin accumulation and higher expression level of anthocyanin biosynthetic genes in both petunia and tobacco. And then, ZeMYB9 was demonstrated to interact with ZeGL3, a bHLH transcription factor belonging to IIIf subgroup. Promoter activity of ZeF3'H was significantly promoted by co-expressing ZeMYB9 and ZeGL3 compared with expressing ZeMYB9 alone. Moreover, transient co-expression of ZeMYB9 and ZeGL3 induced anthocyanin accumulation in tobacco leaves. Our results suggest that ZeMYB9 could enhance cyanidin synthesis and regulate petal color in Z. elegans though activating the expression of ZeF3'H, by itself or interacting with ZeGL3.

Comprehensive Genomic Survey, Structural Classification, and Expression Analysis of WRKY Transcription Factor Family in Rhododendron simsii

(1) Rhododendron is one of the top ten traditional flowers in China, with both high ornamental and economic values. However, with the change of the environment, Rhododendron suffers from various biological stresses. The WRKY transcription factor is a member of the most crucial transcription factor families, which plays an essential regulatory role in a variety of physiological processes and developmental stresses. (2) In this study, 57 RsWRKYs were identified using genome data and found to be randomly distributed on 13 chromosomes. Based on gene structure and phylogenetic relationships, 57 proteins were divided into three groups: I, II, and III. Multiple alignments of RsWRKYs with Arabidopsis thaliana homologous genes revealed that WRKY domains in different groups had different conserved sites. RsWRKYs have a highly conserved domain, WRKYGQK, with three variants, WRKYGKK, WRKYGEK, and WRKYGRK. Furthermore, cis-acting elements analysis revealed that all of the RsWRKYs had stress and plant hormone cis-elements, with figures varying by group. Finally, the expression patterns of nine WRKY genes treated with gibberellin acid (GA), methyl jasmonate (MeJA), heat, and drought in Rhododendron were also measured using quantitative real-time PCR (qRT-PCR). The results showed that the expression levels of the majority of RsWRKY genes changed in response to multiple phytohormones and abiotic stressors. (3) This current study establishes a theoretical basis for future studies on the response of RsWRKY transcription factors to various hormone and abiotic stresses as well as a significant foundation for the breeding of new stress-tolerant Rhododendron varieties.

Combined de novo transcriptomic and physiological analyses reveal RyALS3-mediated aluminum tolerance in Rhododendron yunnanense Franch.

Rhododendron (Ericaceae) not only has ornamental value, but also has great medicinal and edible values. Many Rhododendron species are native to acid soils where aluminum (Al) toxicity limits plant productivity and species distribution. However, it remains unknown how Rhododendron adapts to acid soils. Here, we investigated the physiological and molecular mechanisms of Al tolerance in Rhododendron yunnanense Franch. We found that the shoots of R. yunnanense Franch did not accumulate Al after exposure of seedlings to 50 µM Al for 7 days but predominantly accumulated in roots, suggesting that root Al immobilization contributes to its high Al tolerance. Whole-genome de novo transcriptome analysis was carried out for R. yunnanense Franch root apex in response to 6 h of 50 µM Al stress. A total of 443,639 unigenes were identified, among which 1,354 and 3,413 were up- and down-regulated, respectively, by 6 h of 50 µM Al treatment. Both Gene Ontology (GO) enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that genes involved in "ribosome" and "cytoskeleton" are overrepresented. Additionally, we identified Al-tolerance homologous genes including a tonoplast-localized ABC transporter RyALS3; 1. Overexpression of RyALS3; 1 in tobacco plants confers transgenic plants higher Al tolerance. However, root Al content was not different between wild-type plants and transgenic plants, suggesting that RyALS3; 1 is responsible for Al compartmentalization within vacuoles. Taken together, integrative transcriptome, physiological, and molecular analyses revealed that high Al tolerance in R. yunnanense Franch is associated with ALS3; 1-mediated Al immobilization in roots.

Distinct biological ages of organs and systems identified from a multi-omics study.

Biological age (BA) has been proposed to evaluate the aging status instead of chronological age (CA). Our study shows evidence that there might be multiple "clocks" within the whole-body system: systemic aging drivers/clocks overlaid with organ/tissue-specific counterparts. We utilize multi-omics data, including clinical tests, immune repertoire, targeted metabolomic molecules, gut microbiomes, physical fitness examinations, and facial skin examinations, to estimate the BA of different organs (e.g., liver, kidney) and systems (immune and metabolic system). The aging rates of organs/systems are diverse. People's aging patterns are different. We also demonstrate several applications of organs/systems BA in two independent datasets. Mortality predictions are compared among organs' BA in the dataset of the United States National Health and Nutrition Examination Survey. Polygenic risk score of BAs constructed in the Chinese Longitudinal Healthy Longevity Survey cohort can predict the possibility of becoming centenarian.

Genome-Wide Identification of Petunia HSF Genes and Potential Function of PhHSF19 in Benzenoid/Phenylpropanoid Biosynthesis.

Volatile benzenoids/phenylpropanoids are the main flower scent compounds in petunia (Petunia hybrida). Heat shock factors (HSFs), well known as the main regulator of heat stress response, have been found to be involved in the biosynthesis of benzenoid/phenylpropanoid and other secondary metabolites. In order to figure out the potential function of HSFs in the regulation of floral scent in petunia, we systematically identified the genome-wide petunia HSF genes and analyzed their expression and then the interaction between the key petunia HSF gene with target gene involved in benzenoid/phenylpropanoid biosynthesis. The results revealed that 34 HSF gene family members were obtained in petunia, and most petunia HSFs contained one intron. The phylogenetic analysis showed that 23 petunia HSFs were grouped into the largest subfamily HSFA, while only two petunia HSFs were in HSFC subfamily. The DBD domain and NLS motif were well conserved in most petunia HSFs. Most petunia HSF genes' promoters contained STRE motifs, the highest number of cis-acting element. PhHSF19 is highly expressed in petal tubes, followed by peduncles and petal limbs. During flower development, the expression level of PhHSF19 was dramatically higher at earlier flower opening stages than that at the bud stage, suggesting that PhHSF19 may have potential roles in regulating benzenoid/phenylpropanoid biosynthesis. The expression pattern of PhHSF19 is positively related with PhPAL2, which catalyzes the first committed step in the phenylpropanoid pathway. In addition, there are three STRE elements in the promoter of PhPAL2. PhHSF19 was proven to positively regulate the expression of PhPAL2 according to the yeast one hybrid and dual luciferase assays. These results lay a theoretical foundation for further studies of the regulation of HSFs on plant flower scent biosynthesis.

Corrigendum: Germline-Encoded TCR-MHC Contacts Promote TCR V Gene Bias in Umbilical Cord Blood T Cell Repertoire.

[This corrects the article DOI: 10.3389/fimmu.2019.02064.].

Developing an Unbiased Multiplex PCR System to Enrich the TRB Repertoire Toward Accurate Detection in Leukemia.

Accurate T cell receptor repertoire profiling has provided novel biological and clinical insights in widespread immunological settings; however, there is a lack of reference materials in the community that can be used to calibrate and optimize the various experimental systems in different laboratories. In this study, we designed and synthesized 611 T cell receptor (TCR) beta chain (TRB) templates and used them as reference materials to optimize the multiplex PCR experimental system to enrich the TRB repertoire. We assessed the stability of the optimized system by repeating the experiments in different batches and by remixing the TRB templates in different ratios. These TRB reference materials could be used as independent positive controls to assess the accuracy of the experimental system, and they can also be used as spike-in materials to calibrate the residual biases of the experimental system. We then used the optimized system to detect the minimal residual disease of T cell acute lymphoblastic leukemia and showed a higher sensitivity compared with flow cytometry. We also interrogated how chemotherapy affected the TCR repertoire of patients with B-cell acute lymphoblastic leukemia. Our result shows that high-avidity T cells, such as those targeting known pathogens, are largely selected during chemotherapy, despite the global immunosuppression. These T cells were stimulated and emerged at the time of induction treatment and further expanded during consolidation treatment, possibly to fight against infections. These data demonstrate that accurate immune repertoire information can improve our understanding of the adaptive immunity in leukemia and lead to better treatment management of the patients.

Single-cell RNA-seq unveils critical regulators of human FOXP3+ regulatory T cell stability.

The heterogeneity and plasticity of T lymphocytes is critical for determining immune response outcomes. Functional regulatory T (Treg) cells are commonly characterized by stable FOXP3 expression and have reported to exhibit heterogeneous phenotypes under inflammatory conditions. However, the interplay between inflammation and Treg cell suppressive activity still remains elusive. Here, we utilized single-cell RNA sequencing to investigate how human Treg cells respond to the pro-inflammatory cytokine interleukin-6 (IL-6). We observed that Treg cells divided into two subpopulations after IL-6 stimulation. TIGIT- unstable Treg cells lost FOXP3 expression and gained an effector-like T cell phenotype, whereas TIGIT+ Treg cells retained robust suppressive function. Single cell transcriptome analysis revealed a spectrum of cellular states of IL-6-stimulated Treg cells and how cytochrome P450 family 1 subfamily A member 1 (CYP1A1) is a crucial regulator of Treg cell suppressive capability and stability. CYP1A1-deficient human Treg cells developed a Th17-like phenotype after IL-6 stimulation. Our findings implicate CYP1A1 as a previously unidentified regulator of Treg cells that may have target potential for clinical application for biotherapies.

Germline-Encoded TCR-MHC Contacts Promote TCR V Gene Bias in Umbilical Cord Blood T Cell Repertoire.

T cells recognize antigens as peptides bound to major histocompatibility complex (MHC) proteins through T cell receptors (TCRs) on their surface. To recognize a wide range of pathogens, each individual possesses a substantial number of TCRs with an extremely high degree of variability. It remains controversial whether germline-encoded TCR repertoire is shaped by MHC polymorphism and, if so, what is the preference between MHC genetic variants and TCR V gene compatibility. To investigate the "net" genetic association between MHC variations and TRBV genes, we applied quantitative trait locus (QTL) mapping to test the associations between MHC polymorphism and TCR ß chain V (TRBV) genes usage using umbilical cord blood (UCB) samples of 201 Chinese newborns. We found TRBV gene and MHC loci that are predisposed to interact with one another differ from previous conclusions. The majority of MHC amino acid residues associated with the TRBV gene usage show spatial proximities in known structures of TCR-pMHC complexes. These results show for the first time that MHC variants bias TRBV gene usage in UCB of Chinese ancestry and indicate that germline-encoded contacts influence TCR-MHC interactions in intact T cell repertoires.

Single-cell transcriptomic landscape of nucleated cells in umbilical cord blood.

BACKGROUND: For both pediatric and adult patients, umbilical cord blood (UCB) transplant is a therapeutic option for a variety of hematologic diseases, such as blood cancers, myeloproliferative disorders, genetic diseases, and metabolic disorders. However, the level of cellular heterogeneity and diversity of nucleated cells in UCB has not yet been assessed in an unbiased and systemic fashion. In the present study, nucleated cells from UCB were subjected to single-cell RNA sequencing to simultaneously profile the gene expression signatures of thousands of cells, generating a rich resource for further functional studies. Here, we report the transcriptomes of 17,637 UCB cells, covering 12 major cell types, many of which can be further divided into distinct subpopulations. RESULTS: Pseudotemporal ordering of nucleated red blood cells identifies wave-like activation and suppression of transcription regulators, leading to a polarized cellular state, which may reflect nucleated red blood cell maturation. Progenitor cells in UCB also comprise 2 subpopulations with activation of divergent transcription programs, leading to specific cell fate commitment. Detailed profiling of cytotoxic cell populations unveiled granzymes B and K signatures in natural killer and natural killer T-cell types in UCB. CONCLUSIONS: Taken together, our data form a comprehensive single-cell transcriptomic landscape that reveals previously unrecognized cell types, pathways, and mechanisms of gene expression regulation. These data may contribute to the efficacy and outcome of UCB transplant, broadening the scope of research and clinical innovations.

T cell receptor ß repertoires as novel diagnostic markers for systemic lupus erythematosus and rheumatoid arthritis.

OBJECTIVE: T cell receptor (TCR) diversity determines the autoimmune responses in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and is closely associated with autoimmune diseases prognosis and prevention. However, the characteristics of variations in TCR diversity and their clinical significance is still unknown. Large series of patients must be studied in order to elucidate the effects of these variations. METHODS: Peripheral blood from 877 SLE patients, 206 RA patients and 439 healthy controls (HC) were amplified for the TCR repertoire and sequenced using a high-throughput sequencer. We have developed a statistical model to identify disease-associated TCR clones and diagnose autoimmune diseases. RESULTS: Significant differences were identified in variable (V), joining (J) and V-J pairing between the SLE or RA and HC groups. These differences can be utilised to discriminate the three groups with perfect accuracy (V: area under receiver operating curve > 0.99). One hundred ninety-eight SLE-associated and 53 RA-associated TCRs were identified and used for diseases classification by cross validation with high specificity and sensitivity. Disease-associated clones showed common features and high similarity between both autoimmune diseases. SLE displayed higher TCR heterogeneity than RA with several organ specific properties. Furthermore, the association between clonal expansion and the concentration of disease-associated clones with disease severity were identified, and pathogen-related TCRs were enriched in both diseases. CONCLUSIONS: These characteristics of the TCR repertoire, particularly the disease-associated clones, can potentially serve as biomarkers and provide novel insights for disease status and therapeutical targets in autoimmune diseases.