Genome-wide identification, classification and expression analysis of the heat shock transcription factor family in Garlic (Allium sativum L.).

BACKGROUND: The heat shock transcription factor (HSF) plays a crucial role in the regulatory network by coordinating responses to heat stress as well as other stress signaling pathways. Despite extensive studies on HSF functions in various plant species, our understanding of this gene family in garlic, an important crop with nutritional and medicinal value, remains limited. In this study, we conducted a comprehensive investigation of the entire garlic genome to elucidate the characteristics of the AsHSF gene family. RESULTS: In this study, we identified a total of 17 AsHSF transcription factors. Phylogenetic analysis classified these transcription factors into three subfamilies: Class A (9 members), Class B (6 members), and Class C (2 members). Each subfamily was characterized by shared gene structures and conserved motifs. The evolutionary features of the AsHSF genes were investigated through a comprehensive analysis of chromosome location, conserved protein motifs, and gene duplication events. These findings suggested that the evolution of AsHSF genes is likely driven by both tandem and segmental duplication events. Moreover, the nucleotide diversity of the AsHSF genes decreased by only 0.0002% from wild garlic to local garlic, indicating a slight genetic bottleneck experienced by this gene family during domestication. Furthermore, the analysis of cis-acting elements in the promoters of AsHSF genes indicated their crucial roles in plant growth, development, and stress responses. qRT-PCR analysis, co-expression analysis, and protein interaction prediction collectively highlighted the significance of Asa6G04911. Subsequent experimental investigations using yeast two-hybridization and yeast induction experiments confirmed its interaction with HSP70/90, reinforcing its significance in heat stress. CONCLUSIONS: This study is the first to unravel and analyze the AsHSF genes in garlic, thereby opening up new avenues for understanding their functions. The insights gained from this research provide a valuable resource for future investigations, particularly in the functional analysis of AsHSF genes.

Dynamic network biomarker analysis discovers IbNAC083 in the initiation and regulation of sweet potato root tuberization.

The initiation and development of storage roots (SRs) are intricately regulated by a transcriptional regulatory network. One key challenge is to accurately pinpoint the tipping point during the transition from pre-swelling to SRs and to identify the core regulators governing such a critical transition. To solve this problem, we performed a dynamic network biomarker (DNB) analysis of transcriptomic dynamics during root development in Ipomoea batatas (sweet potato). First, our analysis identified stage-specific expression patterns for a significant proportion (>9%) of the sweet potato genes and unraveled the chronology of events that happen at the early and later stages of root development. Then, the results showed that different root developmental stages can be depicted by co-expressed modules of sweet potato genes. Moreover, we identified the key components and transcriptional regulatory network that determine root development. Furthermore, through DNB analysis an early stage, with a root diameter of 3.5 mm, was identified as the critical period of SR swelling initiation, which is consistent with morphological and metabolic changes. In particular, we identified a NAM/ATAF/CUC (NAC) domain transcription factor, IbNAC083, as a core regulator of this initiation in the DNB-associated network. Further analyses and experiments showed that IbNAC083, along with its associated differentially expressed genes, induced dysfunction of metabolism processes, including the biosynthesis of lignin, flavonol and starch, thus leading to the transition to swelling roots.

Starch synthase II plays a crucial role in starch biosynthesis and the formation of multienzyme complexes in cassava storage roots.

Starch is a glucose polymer synthesized by green plants for energy storage and is crucial for plant growth and reproduction. The biosynthesis of starch polysaccharides is mediated by members of the large starch synthase (SS) protein superfamily. Here, we showed that in cassava storage roots, soluble starch synthase II (MeSSII) plays an important role in starch biosynthesis and the formation of protein complexes with other starch biosynthetic enzymes by directly interacting with MeSSI, MeSBEII, and MeISAII. MeSSII-RNAi cassava lines showed increased amylose content and reduced biosynthesis of the intermediate chain of amylopectin (B1 type) in their storage roots, leading to altered starch physicochemical properties. Furthermore, gel permeation chromatography analysis of starch biosynthetic enzymes between wild type and MeSSII-RNAi lines confirmed the key role of MeSSII in the organization of heteromeric starch synthetic protein complexes. The lack of MeSSII in cassava also reduced the capacity of MeSSI, MeSBEII, MeISAI, and MeISAII to bind to starch granules. These findings shed light on the key components of the starch biosynthesis machinery in root crops.

Genome-wide identification, phylogeny and expression analysis of AP2/ERF transcription factors family in sweet potato.

BACKGROUND: In recent years, much attention has been given to AP2/ERF transcription factors because they play indispensable roles in many biological processes, such as plant development and biotic and abiotic stress responses. Although AP2/ERFs have been thoroughly characterised in many plant species, the knowledge about this family in the sweet potato, which is a vital edible and medicinal crop, is still limited. In this study, a comprehensive genome-wide investigation was conducted to characterise the AP2/ERF gene family in the sweet potato. RESULTS: Here, 198 IbAP2/ERF transcription factors were obtained. Phylogenetic analysis classified the members of the IbAP2/ERF family into three groups, namely, ERF (172 members), AP2 (21 members) and RAV (5 members), which was consistent with the analysis of gene structure and conserved protein domains. The evolutionary characteristics of these IbAP2/ERF genes were systematically investigated by analysing chromosome location, conserved protein motifs and gene duplication events, indicating that the expansion of the IbAP2/ERF gene family may have been caused by tandem duplication. Furthermore, the analysis of cis-acting elements in IbAP2/ERF gene promoters implied that these genes may play crucial roles in plant growth, development and stress responses. Additionally, the available RNA-seq data and quantitative real-time PCR (qRT-PCR) were used to investigate the expression patterns of IbAP2/ERF genes during sweet potato root development as well as under multiple forms of abiotic stress, and we identified several developmental stage-specific and stress-responsive IbAP2/ERF genes. Furthermore, g59127 was differentially expressed under various stress conditions and was identified as a nuclear protein, which was in line with predicted subcellular localization results. CONCLUSIONS: This study originally revealed the characteristics of the IbAP2/ERF superfamily and provides valuable resources for further evolutionary and functional investigations of IbAP2/ERF genes in the sweet potato.

Production of very-high-amylose cassava by post-transcriptional silencing of branching enzyme genes.

High amylose starch can be produced by plants deficient in the function of branching enzymes (BEs). Here we report the production of transgenic cassava (Manihot esculenta Crantz) with starches containing up to 50% amylose due to the constitutive expression of hair-pin dsRNAs targeting the BE1 or BE2 genes. All BE1-RNAi plant lines (BE1i) and BE2-RNAi plant lines (BE2i) were grown up in the field, but with reduced total biomass production. Considerably high amylose content in the storage roots of BE2i plant lines was achieved. Storage starch granules of BE1i and BE2i plants had similar morphology as wild type (WT), however, the size of BE1i starch granules were bigger than that of WT. Comparisons of amylograms and thermograms of all three sources of storage starches revealed dramatic changes to the pasting properties and a higher melting temperature for BE2i starches. Glucan chain length distribution analysis showed a slight increase in chains of DP>36 in BE1i lines and a dramatic increase in glucan chains between DP 10-20 and DP>40 in BE2i lines. Furthermore, BE2i starches displayed a B-type X-ray diffraction pattern instead of the A-type pattern found in BE1i and WT starches. Therefore, cassava BE1 and BE2 function differently in storage root starch biosynthesis.

Genome-wide identification, characterization and expression analysis of the bZIP transcription factors in garlic (Allium sativum L.).

Introduction: The bZIP genes (bZIPs) are essential in numerous biological processes, including development and stress responses. Despite extensive research on bZIPs in many plants, a comprehensive genome-wide analysis of bZIPs in garlic has yet to be undertaken. Methods: In this study, we identified and classified 64 AsbZIP genes (AsbZIPs) into 10 subfamilies. A systematic analysis of the evolutionary characteristics of these AsbZIPs, including chromosome location, gene structure, conserved motifs, and gene duplication, was conducted. Furthermore, we also examined the nucleotide diversity, cis-acting elements, and expression profiles of AsbZIPs in various tissues and under different abiotic stresses and hormone treatments. Results and Discussion: Our findings revealed that gene replication plays a crucial role in the expansion of AsbZIPs, with a minor genetic bottleneck observed during domestication. Moreover, the identification of cis-acting elements suggested potential associations of AsbZIPs with garlic development, hormone, and stress responses. Several AsbZIPs exhibited tissue-preferential and stress/hormone-responsive expression patterns. Additionally, Asa7G01972 and Asa7G01379 were notably differentially expressed under various stresses and hormone treatments. Subsequent yeast two-hybridization and yeast induction experiments validated their interactions with Asa1G01577, a homologue of ABI5, reinforcing their importance in hormone and abiotic stress responses. This study unveiled the characteristics of the AsbZIP superfamily and lays a solid foundation for further functional analysis of AsbZIP in garlic.

DNA methylation profiling to determine the primary sites of metastatic cancers using formalin-fixed paraffin-embedded tissues.

Identifying the primary site of metastatic cancer is critical to guiding the subsequent treatment. Approximately 3-9% of metastatic patients are diagnosed with cancer of unknown primary sites (CUP) even after a comprehensive diagnostic workup. However, a widely accepted molecular test is still not available. Here, we report a method that applies formalin-fixed, paraffin-embedded tissues to construct reduced representation bisulfite sequencing libraries (FFPE-RRBS). We then generate and systematically evaluate 28 molecular classifiers, built on four DNA methylation scoring methods and seven machine learning approaches, using the RRBS library dataset of 498 fresh-frozen tumor tissues from primary cancer patients. Among these classifiers, the beta value-based linear support vector (BELIVE) performs the best, achieving overall accuracies of 81-93% for identifying the primary sites in 215 metastatic patients using top-k predictions (k = 1, 2, 3). Coincidentally, BELIVE also successfully predicts the tissue of origin in 81-93% of CUP patients (n = 68).

DNA methylation and expression profiles of placenta and umbilical cord blood reveal the characteristics of gestational diabetes mellitus patients and offspring.

BACKGROUND: Gestational diabetes mellitus (GDM) is a common pregnancy-specific disease and is growing at an alarming rate worldwide, which can negatively affect the health of pregnant women and fetuses. However, most studies are limited to one tissue, placenta or umbilical cord blood, usually with one omics assay. It is thus difficult to systematically reveal the molecular mechanism of GDM and the key influencing factors on pregnant women and offspring. RESULTS: We recruited a group of 21 pregnant women with GDM and 20 controls without GDM. For each pregnant woman, reduced representation bisulfite sequencing and RNA-seq were performed using the placenta and paired neonatal umbilical cord blood specimens. Differentially methylated regions (DMRs) and differentially expressed genes (DEGs) were identified with body mass index as a covariate. Through the comparison of GDM and control samples, 2779 and 141 DMRs, 1442 and 488 DEGs were identified from placenta and umbilical cord blood, respectively. Functional enrichment analysis showed that the placenta methylation and expression profiles of GDM women mirrored the molecular characteristics of "type II diabetes" and "insulin resistance." Methylation-altered genes in umbilical cord blood were associated with pathways "type II diabetes" and "cholesterol metabolism." Remarkably, both DMRs and DEGs illustrated significant overlaps among placenta and umbilical cord blood samples. The overlapping DMRs were associated with "cholesterol metabolism." The top-ranking pathways enriched in the shared DEGs include "growth hormone synthesis, secretion and action" and "type II diabetes mellitus." CONCLUSIONS: Our research demonstrated the epigenetic and transcriptomic alternations of GDM women and offspring. Our findings emphasized the importance of epigenetic modifications in the communication between pregnant women with GDM and offspring, and provided a reference for the prevention, control, treatment, and intervention of perinatal deleterious events of GDM and neonatal complications.

Coevolutionary insights between promoters and transcription factors in the plant and animal kingdoms.

The divergence and continuous evolution of plants and animals contribute to ecological diversity. Promoters and transcription factors (TFs) are key determinants of gene regulation and transcription throughout life. However, the evolutionary trajectories and relationships of promoters and TFs are still poorly understood. Here, we conducted extensive analysis of large-scale multi-omics sequences in 420 animal species and 223 plant species spanning nearly a billion years of evolutionary history. Results showed that promoter GC-content and TF isoelectric points, as features/signatures that accompany long biological evolution, exhibited increasing growth in animal cells but a decreasing trend in plant cells. Furthermore, the evolutionary trajectories of promoter and TF signatures in the animal kingdom provided further evidence that Mammalia as well as Aves evolved directly from the ancestor Reptilia. The strong correlation between promoter and TF signatures indicates that promoters and TFs formed antagonistic coevolution in the animal kingdom, but mutualistic coevolution in the plant kingdom. The distinct coevolutionary patterns potentially drive the plant-animal divergence，divergent evolution and ecological diversity.

In vivo genome-wide CRISPR screening in murine acute myeloid leukemia uncovers microenvironmental dependencies.

Genome-wide CRISPR screens have been extremely useful in identifying therapeutic targets in diverse cancers by defining genes that are essential for malignant growth. However, most CRISPR screens were performed in vitro and thus cannot identify genes that are essential for interactions with the microenvironment in vivo. Here, we report genome-wide CRISPR screens in 2 in vivo murine models of acute myeloid leukemia (AML) driven by the KMT2A/MLLT3 fusion or by the constitutive coexpression of Hoxa9 and Meis1. Secondary validation using a focused library identified 72 genes specifically essential for leukemic growth in vivo, including components of the major histocompatibility complex class I complex, Cd47, complement receptor Cr1l, and the ß-4-galactosylation pathway. Importantly, several of these in vivo-specific hits have a prognostic effect or are inferred to be master regulators of protein activity in human AML cases. For instance, we identified Fermt3, a master regulator of integrin signaling, as having in vivo-specific dependency with high prognostic relevance. Overall, we show an experimental and computational pipeline for genome-wide functional screens in vivo in AML and provide a genome-wide resource of essential drivers of leukemic growth in vivo.

Alpha-Glucan, Water Dikinase 1 Affects Starch Metabolism and Storage Root Growth in Cassava (Manihot esculenta Crantz).

ABSTARCT: Regulation of storage root development by source strength remains largely unknown. The cassava storage root delay (srd) T-DNA mutant postpones storage root development but manifests normal foliage growth as wild-type plants. The SRD gene was identified as an orthologue of α-glucan, water dikinase 1 (GWD1), whose expression is regulated under conditions of light/dark cycles in leaves and is associated with storage root development. The GWD1-RNAi cassava plants showed both retarded plant and storage root growth, as a result of starch excess phenotypes with reduced photosynthetic capacity and decreased levels of soluble saccharides in their leaves. These leaves contained starch granules having greatly increased amylose content and type C semi-crystalline structures with increased short chains that suggested storage starch. In storage roots of GWD1-RNAi lines, maltose content was dramatically decreased and starches with much lower phosphorylation levels showed a drastically reduced ß-amylolytic rate. These results suggested that GWD1 regulates transient starch morphogenesis and storage root growth by decreasing photo-assimilation partitioning from the source to the sink and by starch mobilization in root crops.