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Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

To evaluate water-nitrogen interactions on irrigated wheat production, it is important to know: the dose, the fractioning, and the most recommended period of fertilizers application; the best irrigation volumes and schedules for meeting the crop requirements, ensuring the minimum leaching risks, to obtain the highest yields and quality of the grain, while achieving high water and nitrogen use efficiencies. This study evaluated the interactive effect of irrigation and nitrogen fertilization, with conventional and enhanced efficiency fertilizers, on yield, irrigation water use efficiency and grain quality of soft wheat. For this purpose, two trials were carried out during the 2016/2017 in Beja, Alentejo, under two irrigation strategies: D1 (100% of crop evapotranspiration – ETc - throughout the cycle) and D2 (100% of ETc only at four stages: beginning of stem extension; booting; heading; grain filling). In one trial, enhanced efficiency N fertilizers were applied through 6 splitting treatments. In the other, conventional N fertilizer was applied through 5 splitting treatments. The results suggest that, on one hand, early applications of advanced efficiency fertilizers do not compromise the crop yield and, on the other hand, the availability of nitrogen in the booting stage is important to obtain higher grain protein content.

Embryogenesis is supported by dynamic loops of cellular interactions. Here, we create a partial mouse embryo model to elucidate the principles of epiblast (Epi) and extra-embryonic endoderm co-development (XEn). We trigger naive mouse embryonic stem cells to form a blastocyst-stage niche of Epi-like cells and XEn-like cells (3D, hydrogel free and serum free). Once established, these two lineages autonomously progress in minimal medium to form an inner pro-amniotic-like cavity surrounded by polarized Epi-like cells covered with visceral endoderm (VE)-like cells. The progression occurs through reciprocal inductions by which the Epi supports the primitive endoderm (PrE) to produce a basal lamina that subsequently regulates Epi polarization and/or cavitation, which, in return, channels the transcriptomic progression to VE. This VE then contributes to Epi bifurcation into anterior- and posterior-like states. Similarly, boosting the formation of PrE-like cells within blastoids supports developmental progression. We argue that self-organization can arise from lineage bifurcation followed by a pendulum of induction that propagates over time. [ABSTRACT FROM AUTHOR]

Modifications in gene regulation are driving forces in the evolution of organisms. Part of these changes involve cis-regulatory elements (CREs), which contact their target genes through higher-order chromatin structures. However, how such architectures and variations in CREs contribute to transcriptional evolvability remains elusive. We use Hoxd genes as a paradigm for the emergence of regulatory innovations, as many relevant enhancers are located in a regulatory landscape highly conserved in amniotes. Here, we analysed their regulation in murine vibrissae and chicken feather primordia, two skin appendages expressing different Hoxd gene subsets, and compared the regulation of these genes in these appendages with that in the elongation of the posterior trunk. In the two former structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, probably as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the gene regulation that occurs in the mouse and chicken embryonic trunk partially relies on conserved CREs. A high proportion of these noncoding sequences active in the trunk have functionally diverged between species, suggesting that transcriptional robustness is maintained, despite considerable divergence in enhancer sequences. [ABSTRACT FROM AUTHOR]

The nuclear lamina (NL) consists of a thin meshwork of lamins and associated proteins that lines the inner nuclear membrane (INM). In metazoan nuclei, a large proportion of the genome contacts the NL in broad lamina-associated domains (LADs). Contacts of the NL with the genome are believed to aid the spatial organization of chromosomes and contribute to the regulation of transcription. Here, we will focus on recent insights in the structural organization of the genome at the NL and the role of this organization in the regulation of gene expression. [ABSTRACT FROM AUTHOR]

Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework. [ABSTRACT FROM AUTHOR]

Evo-Devo studies rely on a collection of animal model systems belonging to different phylogenetic branches to try and understand how organisms carrying a similar set of genes and pathways can develop into such a variety of shapes and sizes. The squamate clade, however, has only recently started to receive the attention it deserves in particular due to extreme morphological and metabolic aspects and, consequently, the important insights that it could bring in different fields. The recent sequencing of several squamate genomes as well as the generation of high quality trancriptomes for different snake tissues now provide the necessary tools to complement biological studies. Here, we briefly report on recent work involving developing snake embryos to illustrate their interest to assess vertebrate developmental mechanisms. We also discuss the relevance to use snake species as Evo-Devo model systems and potential ways to cross the important limitations intrinsically associated with developmental and genetic studies of these fascinating animals. [ABSTRACT FROM AUTHOR]

Development of the vertebrate axial skeleton requires the concerted activity of several Hox genes. Among them, Hox genes belonging to the paralog group 10 are essential for the formation of the lumbar region of the vertebral column, owing to their capacity to block rib formation. In this work, we explored the basis for the rib-repressing activity of Hox10 proteins. Because genetic experiments in mice demonstrated that Hox10 proteins are strongly redundant in this function, we first searched for common motifs among the group members. We identified the presence of two small sequences flanking the homeodomain that are phylogenetically conserved among Hox10 proteins and that seem to be specific for this group. We show here that one of these motifs is required but not sufficient for the rib-repressing activity of Hox10 proteins. This motif includes two potential phosphorylation sites, which are essential for protein activity as their mutation to alanines resulted in a total loss of rib-repressing properties. Our data indicates that this motif has a significant regulatory function, modulating interactions with more N-terminal parts of the Hox protein, eventually triggering the rib-repressing program. In addition, this motif might also regulate protein activity by alteration of the protein's DNA-binding affinity through changes in the phosphorylation state of two conserved tyrosine residues within the homeodomain. [ABSTRACT FROM AUTHOR]

Irrigation-induced or secondary salinization can occur when salts are introduced by irrigation water and accumulate within the root zone due to insufficient leaching. Mediterranean regions are especially susceptible, given the predominant climate and the expanding of irrigation areas. In this study, two indices to assess the risk of salinization (RSA) and sodification (RSO), previously applied at a regional scale, were used in a hydro-agricultural area (AHA) in Southern Portugal, in ten crop fields. Information on climate, irrigation water quality, soil characteristics, and land use was obtained from large databases and from local data. The results revealed the feasibility of using the RSA and RSO indices both on large and smaller scales, seeing as most of the area in the monitored crop fields presented the same risk classes (62% in RSA and 78% in RSO). Deviations were due to the reduction in scores for drainage and, in the case of RSO, the assigned irrigation method based on the land occupation class. Considering that different spatial scales of risk assessment are associated with different objectives and management options, a risk management framework was outlined following a multi-scale perspective for mitigation actions in salt-sensitive areas, ranging from territorial planning to the adoption of on-farm practices that can contribute to the sustainability of irrigated agriculture. [ABSTRACT FROM AUTHOR]

Recent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, but the combined detection of histone post-translational modifications and transcription at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of single-chain variable fragment antibodies, engineered chromatin reader domains, and endogenous chromatin-binding proteins. Using these, we render the DamID technology compatible with the genome-wide identification of histone post-translational modifications. Importantly, this includes the possibility to jointly measure chromatin marks and transcription at the single-cell level. We use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. In addition, we map H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specific to notochord. Overall, EpiDamID is a new addition to a vast toolbox to study chromatin states during dynamic cellular processes. [Display omitted] • EpiDamID extends the use of DamID-based protocols to epigenetic chromatin marks • Histone PTM-specific binding domains target the Dam enzyme to the mark of interest • A single-cell implementation offers joint epigenetic and transcriptomic readouts Rang and de Luca et al. develop EpiDamID, an addition to the DamID toolkit that enables the study of histone post-translational modifications. EpiDamID can be implemented in various biological systems and methodological approaches, including simultaneous single-cell measurement of epigenetic state and transcription during embryogenesis. [ABSTRACT FROM AUTHOR]

The effect of regulated deficit irrigation (RDI) strategies on fruit yield and quality at harvest and during cold storage at 5 °C of Wonderful pomegranates was studied. Three irrigation strategies were tested: control strategy (C) using farm irrigation with 100% of crop evaporation (ETc), and two RDI strategies where 25% of ETc was applied during flowering and fruit set (RDI 1) and during the final phase of fruit growth and ripening (RDI 2). The RDI 1 approach reduced the pomegranate yield. Conversely, the RDI 2 allowed a similar pomegranate yield to C treatment. Fruit morphological characteristics were not affected by RDI at harvest and during cold storage. Both RDI strategies resulted in fruits with higher aril percentages. Physicochemical parameters of fruits at harvest and during cold storage were not affected by irrigation strategies, except the total soluble solids content that was higher in fruits from RDI 2 than in those from C and RDI 1. During storage, a reduction of the fruit weight and size was observed, particularly in the first 15 days of storage. The total soluble solids content and titratable acidity decrease, while pH increases with storage. At harvest and during cold storage, the total phenolic content and antioxidant activity evaluated by ABTS+ assay did not differ among irrigation treatments, whereas using a FRAP assay fruits from RDI trended to have higher antioxidant activity than those from C treatment. Total phenolic content and ABTS+ values were lower at the final of storage time than on the first day. Present results showed that RDI strategies applied in a final phase of fruit growth and ripening could be a convenient irrigation strategy for commercial Wonderful orchards in the south of Portugal, allowing improve the fruit quality with a water saving of 14.6%. • Regular deficit irrigation improved water saving on pomegranate orchards. • Irrigation water saving during ripening did not affect the pomegranate yield. • Pomegranate yield decreased with water restriction during flowering-fruit set. • Irrigation water restriction during ripening increased arils soluble solids content. • Regular deficit irrigation did not affect the fruit attributes during cold storage. [ABSTRACT FROM AUTHOR]