The dynamic proteome in Arabidopsis thaliana early embryogenesis.

The morphology of the flowering plant is established during early embryogenesis. In recent years, many studies have focused on transcriptional profiling in plant embryogenesis, but the dynamic landscape of the Arabidopsis thaliana proteome remains elusive. In this study, Arabidopsis embryos at 2/4-cell, 8-cell, 16-cell, 32-cell, globular and heart stages were collected for nanoproteomic analysis. In total, 5386 proteins were identified. Of these, 1051 proteins were universally identified in all developmental stages and a range of 27 to 2154 proteins was found to be stage specific. These proteins could be grouped into eight clusters according to their expression levels. Gene Ontology enrichment analysis showed that genes involved in ribosome biogenesis and auxin-activated signalling were enriched during early embryogenesis, indicating that active translation and auxin signalling are important events in Arabidopsis embryo development. Combining RNA-sequencing data with the proteomics analysis, the correlation between mRNA and protein was evaluated. An overall positive correlation was found between mRNA and protein. This work provides a comprehensive landscape of the Arabidopsis proteome in early embryogenesis. Some important proteins/transcription factors identified through network analysis may serve as potential targets for future investigation.

Arsenic induces hepatotoxicity in chickens via PANoptosis pathway.

Environmental pollution caused by arsenic or its compounds is called arsenic pollution. Arsenic pollution mainly comes from people's mining and smelting of arsenic compounds. In addition, the widespread use of arsenic compounds, such as the use and production of arsenic-containing pesticides, is also a source of arsenic contamination. Arsenic contamination leads to an increased risk of arsenic exposure, and the multi-organ toxicity induced by arsenic exposure is a global health problem. As a non-mammalian vertebrate with high nutrient levels, chickens readily absorb and accumulate arsenic from their food. Relevant studies have shown that arsenic exposure induces hepatotoxicity in chickens, and there has been a steady stream of research into the specific mechanisms involved. PANoptosis, a newly discovered and unique mode of programmed cell death (PCD) characterized by both apoptosis, cellular pyroptosis, and necroptosis. There are no studies to indicate whether chicken liver toxicity due to arsenic is associated with PANoptosis. Therefore, we established chicken animal models and chicken primary hepatocyte models exposed to different arsenic concentrations to dissect the role and mechanism of PANoptosis in arsenic exposure-induced hepatotoxicity in chickens. Our histopathological results showed that arsenic treatment caused dose-dependent damage to chicken liver structure. Meanwhile, different doses of arsenic treatment groups caused significant up-regulation of the protein level of ZBP1, a key factor of PANoptosis. And then consequently triggered the abnormal gene and protein expression levels of apoptosis-associated factors (Caspase-8, Caspase-7, Caspase-3), cellular pyroptosis-associated factors (NLRP3, ASC, GSDMD) and necroptosis-associated factors (RIPK1, RIPK3, MLKL). In conclusion, our study revealed that PANoptosis is involved in arsenic-induced chicken hepatotoxicity. Our findings provide a new perspective on the pathogenesis of arsenic exposure-induced hepatotoxicity in chickens.

Arsenic-induced mtDNA release promotes inflammatory responses through cGAS-STING signaling in chicken hepatocytes.

Arsenic is a toxic element that can cause severe liver damage in humans and animals. Arsenic-based inorganic pesticides, such as lead arsenate, copper arsenate, and calcium arsenate, are widely used for insect control and can eventually affect human health through accumulation in the food chain. However, the relationship between arsenic trioxide (ATO)-induced hepatotoxicity and the cGAS-STING signaling pathway has not been reported. The aim of this study was to investigate the potential role of inflammatory response in ATO-induced hepatotoxicity in chickens. In this study, we found that ATO exposure resulted in mtDNA leakage into the cytoplasm of chicken hepatocytes, which activated the cGAS-STING pathway and significantly increased the cGAS, STING, TBK1, and IRF7 mRNA and protein expression levels. Moreover, type I interferon response was activated. Concurrently, STING triggered the activation of the traditional NF-κB signaling pathway and promoted the expression of pro-inflammatory cytokine genes, including TNF-α, IL-6, and IL-1ß. Subsequently, we found that both mtDNA clearance with EtBr and inhibition of the cGAS-STING pathway with H-151 reversed the ATO-induced innate immune and inflammatory responses. In summary, the above findings indicate that chicken hepatocytes can induce innate immune responses and inflammatory responses via mtDNA-cGAS-STING under ATO-exposure conditions, which is of great significance for further studies on the toxicity mechanism of ATO.

Identification of the accessible chromatin regions in six tissues in the soybean.

Accessible chromatin regions (ACRs) are tightly associated with gene expressions in the genome. Conserved non-coding cis-regulatory elements, such as transcription factor binding motifs, are usually found in ACRs, indicating an essential regulatory role of ACRs in the plant genome architecture. However, there have been few studies on soybean ACRs, especially those focusing on specific tissues. Hence, in this study, with the convenient ATAC-seq, we identified the ACRs in six soybean tissues, including root, leaf bud, flower, flower bud, developing seed, and pod. In total, the ACRs occupied about 3.3% of the entire soybean genome. By integrating the results from RNA-seq and transcription factor (TF) ChIP-seq, ACRs were found to be tightly associated with gene expressions and TF binding capacities in soybean. Together, these data provide a comprehensive understanding of the genomic features of ACRs in soybean. As a collection of essential genomic resources, these processed data are made available at datahub.wildsoydb.org.

Stress response proteins NRP1 and NRP2 are pro-survival factors that inhibit cell death during ER stress.

Environmental stresses cause an increased number of unfolded or misfolded proteins to accumulate in the endoplasmic reticulum (ER), resulting in ER stress. To restore ER homeostasis and survive, plants initiate an orchestrated signaling pathway known as the unfolded protein response (UPR). Asparagine-rich protein (NRP) 1 and NRP2, two homologous proteins harboring a Development and Cell Death domain, are associated with various stress responses in Arabidopsis (Arabidopsis thaliana), but the relevant molecular mechanism remains obscure. Here, we show that NRP1 and NRP2 act as key pro-survival factors during the ER stress response and that they inhibit cell death. Loss-of-function of NRP1 and NRP2 results in decreased tolerance to the ER stress inducer tunicamycin (TM), accelerating cell death. NRP2 is constitutively expressed while NRP1 is induced in plants under ER stress. In Arabidopsis, basic leucine zipper protein (bZIP) 28 and bZIP60 are important transcription factors in the UPR that activates the expression of many ER stress-related genes. Notably, under ER stress, bZIP60 activates NRP1 by directly binding to the UPRE-I element in the NRP1 promoter. These findings reveal a pro-survival strategy in plants wherein the bZIP60-NRPs cascade suppresses cell death signal transmission, improving survival under adverse conditions.

The gibberellin signaling negative regulator RGA-LIKE3 promotes seed storage protein accumulation.

Seed storage protein (SSP) acts as one of the main components of seed storage reserves, of which accumulation is tightly mediated by a sophisticated regulatory network. However, whether and how gibberellin (GA) signaling is involved in this important biological event is not fully understood. Here, we show that SSP content in Arabidopsis (Arabidopsis thaliana) is significantly reduced by GA and increased in the GA biosynthesis triple mutant ga3ox1/3/4. Further investigation shows that the DELLA protein RGA-LIKE3 (RGL3), a negative regulator of GA signaling, is important for SSP accumulation. In rgl3 and 35S:RGL3-HA, the expression of SSP genes is down- and upregulated, respectively, compared with that in the wild-type. RGL3 interacts with ABSCISIC ACID INSENSITIVE3 (ABI3), a critical transcription factor for seed developmental processes governing SSP accumulation, both in vivo and in vitro, thus greatly promoting the transcriptional activating ability of ABI3 on SSP genes. In addition, genetic evidence shows that RGL3 and ABI3 regulate SSP accumulation in an interdependent manner. Therefore, we reveal a function of RGL3, a little studied DELLA member, as a coactivator of ABI3 to promote SSP biosynthesis during seed maturation stage. This finding advances the understanding of mechanisms in GA-mediated seed storage reserve accumulation.

An expedient survey and characterization of the soybean JAGGED 1 (GmJAG1) transcription factor binding preference in the soybean genome by modified ChIPmentation on soybean protoplasts.

ChIP-seq is widely used for mapping the transcription factor (TF) binding sites throughout the genome in vivo. In this study, we adopted and modified ChIPmentation, a fast, robust, low-input requirement ChIP-seq method, to a transient expression system using soybean protoplasts to expedite the exploration of TF binding sites. To test this new protocol, we expressed a tagged version of a C2H2-type zinc finger TF, JAGGED1 (GmJAG1), in soybean protoplasts and successfully identified its binding sites in the soybean genome. Furthermore, valuable genomic features such as a novel GmJAG1-binding motif, and the epigenetic characteristics as well as an enhancer-like function of GmJBSs were also found via coupling ATAC-seq and H3K27me3 ChIP-seq data. The application of the modified ChIPmentation protocol in this study using soybean protoplasts provided a new approach for rapid elucidation of how a TF binds to the various target genes in the soybean genome, as illustrated here using GmJAG1.

Ectopic expression of Torenia fournieri TCP8 and TCP13 alters the leaf and petal phenotypes in Arabidopsis thaliana.

Teosinte branched1/cycloidea/proliferating cell factor (TCP) transcription factors (TFs) are essential for regulating plant developmental processes, which is still largely unknown in Torenia fournieri (T. fournieri), a widely used horticultural flower. In this study, we used a de novo transcriptome assembly method to predict the TCP transcription factors in T. fournieri. In total, 15 out of 21 predicted T. fournieri TCPs (TfTCPs) were isolated and verified with Sanger sequencing. Phylogenetic analysis showed that these 15 TfTCPs could be classified into two major classes. Most of these TfTCPs were expressed in floral buds, flowers, or leaves, suggesting an important role in developmental regulation in these tissues. Moreover, TfTCP8 and TfTCP13, the homologues of the Arabidopsis thaliana TCP5-like transcription factor, were able to bind to the conserved Class II TCP binding motifs and are localized to the nucleus, indicating that TfTCP8 and TfTCP13 act as transcriptional regulators. In agreement with the overexpression phenotype of AtTCP5, ectopic expression of TfTCP8 and TfTCP13 resulted in narrow leaves and the small petal phenotype in Arabidopsis, suggesting that these two TfTCPs potentially regulate leaf or flower shape in T. fournieri.

Chromatin Accessibility Is Associated with Artemisinin Biosynthesis Regulation in Artemisia annua.

Glandular trichome (GT) is the dominant site for artemisinin production in Artemisia annua. Several critical genes involved in artemisinin biosynthesis are specifically expressed in GT. However, the molecular mechanism of differential gene expression between GT and other tissue types remains elusive. Chromatin accessibility, defined as the degree to which nuclear molecules are able to interact with chromatin DNA, reflects gene expression capacity to a certain extent. Here, we investigated and compared the landscape of chromatin accessibility in Artemisia annua leaf and GT using the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) technique. We identified 5413 GT high accessible and 4045 GT low accessible regions, and these GT high accessible regions may contribute to GT-specific biological functions. Several GT-specific artemisinin biosynthetic genes, such as DBR2 and CYP71AV1, showed higher accessible regions in GT compared to that in leaf, implying that they might be regulated by chromatin accessibility. In addition, transcription factor binding motifs for MYB, bZIP, C2H2, and AP2 were overrepresented in the highly accessible chromatin regions associated with artemisinin biosynthetic genes in glandular trichomes. Finally, we proposed a working model illustrating the chromatin accessibility dynamics in regulating artemisinin biosynthetic gene expression. This work provided new insights into epigenetic regulation of gene expression in GT.

Integrated Dissection of lncRNA-Perturbated Triplets Reveals Novel Prognostic Signatures Across Cancer Types.

Long noncoding RNA (lncRNA)/microRNA(miRNA)/mRNA triplets contribute to cancer biology. However, identifying significative triplets remains a major challenge for cancer research. The dynamic changes among factors of the triplets have been less understood. Here, by integrating target information and expression datasets, we proposed a novel computational framework to identify the triplets termed as "lncRNA-perturbated triplets". We applied the framework to five cancer datasets in The Cancer Genome Atlas (TCGA) project and identified 109 triplets. We showed that the paired miRNAs and mRNAs were widely perturbated by lncRNAs in different cancer types. LncRNA perturbators and lncRNA-perturbated mRNAs showed significantly higher evolutionary conservation than other lncRNAs and mRNAs. Importantly, the lncRNA-perturbated triplets exhibited high cancer specificity. The pan-cancer perturbator OIP5-AS1 had higher expression level than that of the cancer-specific perturbators. These lncRNA perturbators were significantly enriched in known cancer-related pathways. Furthermore, among the 25 lncRNA in the 109 triplets, lncRNA SNHG7 was identified as a stable potential biomarker in lung adenocarcinoma (LUAD) by combining the TCGA dataset and two independent GEO datasets. Results from cell transfection also indicated that overexpression of lncRNA SNHG7 and TUG1 enhanced the expression of the corresponding mRNA PNMA2 and CDC7 in LUAD. Our study provides a systematic dissection of lncRNA-perturbated triplets and facilitates our understanding of the molecular roles of lncRNAs in cancers.

Temporal-Specific Interaction of NF-YC and CURLY LEAF during the Floral Transition Regulates Flowering.

The flowering time of higher plants is controlled by environmental cues and intrinsic signals. In Arabidopsis (Arabidopsis thaliana), flowering is accelerated by exposure to long-day conditions via the key photoperiod-induced factor FLOWERING LOCUS T (FT). Nuclear Factor-Y subunit C (NF-YC) proteins function as important mediators of epigenetic marks in different plant developmental stages and play an important role in the regulation of FT transcription, but the mechanistic details of this remain unknown. In this study, we show that Arabidopsis NF-YC homologs temporally interact with the histone methyltransferase CURLY LEAF (CLF) during the flowering transition. The binding of NF-YC antagonizes the association of CLF with chromatin and the CLF-dependent deposition of H3 lysine-27 trimethylation, thus relieving the repression of FT transcription and facilitating flowering under long-day conditions. Our findings reveal a novel mechanism of NF-YC/CLF-mediated epigenetic regulation of FT activation in photoperiod-induced flowering and, consequently, contribute to our understanding of how plants control developmental events in a temporal-specific regulatory manner.

Phenotypic flexibility of thermogenesis in the hwamei (Garrulax canorus): responses to cold acclimation.

Cold acclimation in birds involves a comprehensive array of physiological and morphological adjustment ranging from changes in aerobic enzyme activity to metabolic rate and organ mass. In the present study, we investigated phenotypic variation in thermogenic activity in the hwamei (Garrulax canorus) under normal (35°C) or cold (15°C) ambient temperature conditions. Acclimation to an ambient temperature of 15°C for 4 wk significantly increased the body mass, basal metabolic rate (BMR), and energy intake, including both gross energy intake and digestible energy intake, compared with birds kept at 35°C. Furthermore, birds acclimated to 15°C increased the dry mass of their liver and kidneys, but not their heart and pectoral muscles, and displayed higher state-4 respiration in the liver, kidneys, heart, and pectoral muscles, and higher cytochrome-c oxidase (COX) activity in liver, kidney, and pectoral muscle, compared with those kept at 35°C. There was a positive correlation between BMR and state-4 respiration in all of the above organs except the liver, and between BMR and COX activity in all of the above organs. Taken together, these data illustrate the morphological, physiological, and enzymatic changes associated with cold acclimation, and support the notion that the hwamei is a bird species from temperate climates that exhibits high phenotypic flexibility of thermogenic capacity.

Seasonal variation in body mass, body temperature and thermogenesis in the Hwamei, Garrulax canorus.

The basal thermogenesis of birds is beginning to be viewed as a highly flexible physiological trait influenced by environmental fluctuations, particularly changes in ambient temperature (Ta). Many birds living in regions with seasonal fluctuations in Ta typically respond to cold by increasing their insulation and adjusting their metabolic rate. To understand these metabolic adaptations, body temperature (Tb), metabolic rate (MR), thermal neutral zone (TNZ) and thermal conductance were measured within a range of temperatures from 5 to 40°C in free-living Hwamei, Garrulax canorus, in both winter and summer. Body mass was 61.2±0.3g in winter and 55.5±1.0g in summer, and mean Tb was 41.6±0.1°C in winter and 42.3±0.1°C in summer. TNZ was between 28.3 and 35.1°C in winter and between 28.7 and 33.2°C in summer. The mean basal metabolic rate (BMR) within TNZ was 203.32±11.81ml O2 h(-1) in winter and 168.99±6.45ml O2 h(-1) in summer. Minimum thermal conductance was 3.73±0.09joulesg(-1)h(-1)°C(-1) in winter and 3.26±0.06joulesg(-1)h(-1)°C(-1) in summer. Birds caught in winter had higher body mass, MR, and more variable TNZ than those in summer. The increased winter BMR indicates improved ability to cope with cold and maintenance of a high Tb. These results show that the Hwamei's metabolism is not constant, but exhibits pronounced seasonal phenotypic flexibility associated with maintenance of a high Tb.

Arsenic trioxide induces innate immune response and inflammatory response in chicken liver via cGAS-STING/NF-κB pathway.

Arsenic is a toxic metal-like element widely used in the pesticide, preservative and semiconductor industries. However, accumulation of arsenic through the food chain can cause serious damage to animal and human health. However, the toxic mechanism of arsenic-induced hepatotoxicity in chickens is not clear, and the present study aimed to investigate the potential role of cGAS-STING and NF-κB pathways on inflammatory injury in chicken liver. In this study, 75 white-feathered broilers were divided into a control group, a low-dose arsenic group (4 mg/kg) and a high-dose arsenic group (8 mg/kg) to investigate the toxic effects of arsenic on chicken liver. In this study, we found that pathological changes such as inflammatory cell infiltration and vesicular degeneration occurred in the liver when exposed to ATO. Crucially, exposure to ATO triggered the cGAS-STING pathway and markedly raised the levels of mRNA and protein expression of cGAS, STING, TBK1, and IRF7. The type I interferon response was also triggered. Simultaneously, STING induced the activation of the conventional NF-κB signaling pathway and stimulated the expression of genes associated with inflammation, such as IL-6, TNF-α and IL-1ß. In summary, the induction of inflammatory responses via cGAS-STING and NF-κB signaling pathways under high ATO exposure provides new ideas for further studies on the toxicological mechanisms of arsenic.

Photoperiod controls plant seed size in a CONSTANS-dependent manner.

Photoperiodic plants perceive changes in day length as seasonal cues to orchestrate their vegetative and reproductive growth. Although it is known that the floral transition of photoperiod-sensitive plants is tightly controlled by day length, how photoperiod affects their post-flowering development remains to be clearly defined, as do the underlying mechanisms. Here we demonstrate that photoperiod plays a prominent role in seed development. We found that long-day (LD) and short-day (SD) plants produce larger seeds under LD and SD conditions, respectively; however, seed size remains unchanged when CONSTANS (CO), the central regulatory gene of the photoperiodic response pathway, is mutated in Arabidopsis and soybean. We further found that CO directly represses the transcription of AP2 (a known regulatory gene of seed development) under LD conditions in Arabidopsis and SD conditions in soybean, thereby controlling seed size in a photoperiod-dependent manner, and that these effects are exerted through regulation of the proliferation of seed coat epidermal cells. Collectively, our findings reveal that a crucial regulatory cascade involving CO-AP2 modulates photoperiod-mediated seed development in plants and provide new insights into how plants with different photoperiod response types perceive seasonal changes that enable them to optimize their reproductive growth.

AaHY5 ChIP-seq based on transient expression system reveals the role of AaWRKY14 in artemisinin biosynthetic gene regulation.

ChIP-seq (Chromatin immunoprecipitation with sequencing) is the gold standard for determining genome-wide in vivo transcription factor binding sites, the first step for targets prediction and network construction. For non-model plants, it is challenging to perform ChIP-seq due to the difficulty in generating stable transgenic plants. AaHY5 is a positive regulator in artemisinin biosynthesis, whose detailed mode of action remains elusive. Here, we established a protoplast transformation procedure for Artemisia annua by optimizing different conditions in protoplast isolation and transfection. We then performed AaHY5 ChIP-seq based on the established transient expression system. Combining RNA-seq data for various tissues, we identified four transcription factors (one MYB and three WRKY family members) in AaHY5 targets that potentially regulated artemisinin biosynthesis. The three WRKY transcription factors could be induced by light and the overexpression of AaHY5 and upregulate two artemisinin biosynthetic genes, ADS and CYP71AV1. Furthermore, AaWRKY14 showed transcriptional activation activity on artemisinin biosynthetic gene CYP71AV1. Together, AaWRKY14 was identified as a potential transcription factor linking AaHY5 and the artemisinin biosynthetic gene regulation.

Genomic Features of Open Chromatin Regions (OCRs) in Wild Soybean and Their Effects on Gene Expressions.

Transcription activation is tightly associated with the openness of chromatin, which allows direct contact between transcriptional regulators, such as transcription factors, and their targeted DNA for downstream gene activation. However, the annotation of open chromatin regions (OCRs) in the wild soybean (Glycine soja) genome is limited. We performed assay for transposase-accessible chromatin using sequencing (ATAC-seq) and successfully identified 22,333 OCRs in the leaf of W05 (a wild soybean accession). These OCRs were enriched in gene transcription start sites (TSS) and were positively correlated with downstream gene expression. Several known transcription factor (TF)-binding motifs were also enriched at the OCRs. A potential regulatory network was constructed using these transcription factors and the OCR-marked genes. Furthermore, by overlapping the OCR distribution with those of histone modifications from chromatin immunoprecipitation followed by sequencing (ChIP-seq), we found that the distribution of the activation histone mark, H3K4me3, but not that of the repressive H3K27me3 mark, was closely associated with OCRs for gene activation. Several putative enhancer-like distal OCRs were also found to overlap with LincRNA-encoding loci. Moreover, our data suggest that homologous OCRs could potentially influence homologous gene expression. Hence, the duplication of OCRs might be essential for plant genome architecture as well as for regulating gene expression.

Gibberellins play an essential role in late embryogenesis of Arabidopsis.

The plant hormone gibberellin plays key roles in almost all aspects of plant development, but its detailed function and underlying regulatory mechanism in embryo development are not yet clearly defined. Here, we illustrate an essential role of gibberellin in late embryogenesis of Arabidopsis. Bioactive gibberellins are highly biosynthesized during the late developmental stage of embryos. At that time, deficiency in gibberellin biosynthesis or signalling results in an abnormal embryo phenotype characterized by less-developed cotyledons and shortened embryo axis. In contrast, gibberellin overdose leads to a significantly larger size of mature embryo. We reveal that the gibberellin signalling repressor DELLA interact with LEAFY COTYLEDON1 (LEC1), the key regulator in late embryogenesis. Gibberellin triggers the degradation of DELLAs to relieve their repression of LEC1, thus promoting auxin accumulation to facilitate embryo development. Therefore, we uncover a space/time-specific role of gibberellin in regulating late embryogenesis through the gibberellin-DELLA-LEC1 signalling cascade, providing a novel mechanistic understanding of how phytohormones regulate embryogenesis.

Effects of temperature acclimation on body mass and energy budget in the Chinese bulbul Pycnonotus sinensis.

Chinese bulbuls (Pycnonotus sinensis) are small passerine birds that inhabit areas of central, southern and eastern China. Previous observations suggest that free-living individuals of this species may change their food intake in response to seasonal changes in ambient temperature. In the present study, we randomly assigned Chinese bulbuls to either a 30℃ or 10℃ group, and measured their body mass (BM), body temperature, gross energy intake (GEI), digestible energy intake (DEI), and the length and mass of their digestive tracts over 28 days of acclimation at these temperatures. As predicted, birds in the 30℃ group had lower body mass, GEI and DEI relative to those in the 10℃ group. The length and mass of the digestive tract was also lower in the 30℃ group and trends in these parameters were positively correlated with BM, GEI and DEI. These results suggest that Chinese bulbuls reduced their absolute energy demands at relatively high temperatures by decreasing their body mass, GEI and DEI, and digestive tract size.