Functional Differentiation of the Duplicated Gene BrrCIPK9 in Turnip (Brassica rapa var. rapa).

Gene duplication is a key biological process in the evolutionary history of plants and an important driving force for the diversification of genomic and genetic systems. Interactions between the calcium sensor calcineurin B-like protein (CBL) and its target, CBL-interacting protein kinase (CIPK), play important roles in the plant's response to various environmental stresses. As a food crop with important economic and research value, turnip (Brassica rapa var. rapa) has been well adapted to the environment of the Tibetan Plateau and become a traditional crop in the region. The BrrCIPK9 gene in turnip has not been characterized. In this study, two duplicated genes, BrrCIPK9.1 and BrrCIPK9.2, were screened from the turnip genome. Based on the phylogenetic analysis, BrrCIPK9.1 and BrrCIPK9.2 were found located in different sub-branches on the phylogenetic tree. Real-time fluorescence quantitative PCR analyses revealed their differential expression levels between the leaves and roots and in response to various stress treatments. The differences in their interactions with BrrCBLs were also revealed by yeast two-hybrid analyses. The results indicate that BrrCIPK9.1 and BrrCIPK9.2 have undergone Asparagine-alanine-phenylalanine (NAF) site divergence during turnip evolution, which has resulted in functional differences between them. Furthermore, BrrCIPK9.1 responded to high-pH (pH 8.5) stress, while BrrCIPK9.2 retained its ancestral function (low K+), thus providing further evidence of their functional divergence. These functional divergence genes facilitate turnip's good adaptation to the extreme environment of the Tibetan Plateau. In summary, the results of this study reveal the characteristics of the duplicated BrrCIPK9 genes and provide a basis for further functional studies of BrrCBLs-BrrCIPKs in turnip.

Morphological and structural characteristics of the elytra reduce impact damage to ladybird beetles.

Beetle elytra act as natural protective covers and effectively shield their flexible abdomens and fragile hindwings from damage. The existing studies have attributed this contribution of the elytra to its honeycomb structures. In this combined experimental and theoretical study, we used the seven-spotted ladybird beetle to demonstrate that both biological morphology and the hollow structure of the dome-like elytra combined to reduce damage during falling. The falling ladybird beetles had a high probability (59.52%) of hitting the ground with the costal edge of the elytra. This strategy could assist with converting the translational energy into rotational kinetic energy, resulting in the reduction of the impulse during falling. In addition, the hollow structures on the elytra could further absorb the residual impact energy. In the future, this biological paradigm could be used as a basis for the development of falling/landing techniques for advanced robots.

Chromosome-level genome assembly of Hippophae gyantsensis.

Hippophae gyantsensis, which is a native tree species in China, is ideal for windbreak and sand-fixing forests. It is an economically and ecologically valuable tree species distributed exclusively in the Qinghai-Tibet Plateau in China. In our study, we assembled a chromosome-level genome of H. gyantsensis using Illumina sequencing, Nanopore sequencing and chromosome structure capture technique. The genome was 716.32 Mb in size with scaffold N50 length of 64.84 Mb. A total of 716.25 Mb genome data was anchored and orientated onto 12 chromosomes with a mounting rate of up to 99.99%. Additionally, the genome was found to comprise approximately 56.84% repeat sequences, of which long terminal repeats(LTRs) that accounted for 33.19% of the entire genome. Meanwhile, a total of 32,316 protein-coding genes were predicted, and 91.07% of these genes were functionally annotated. We also completed a series of comparative genomic analyses to provide researchers with useful reference material for future studies on seabuckthorn.

Animating fossilized invertebrates by motion reconstruction.

Taking the motion reconstruction of the Cretaceous hell ants as an example, this study shows how to achieve motion reconstruction in fossil invertebrates and discusses potential challenges and opportunities.

Chromosome-level genome assembly of Murraya paniculata sheds light on biosynthesis of floral volatiles.

BACKGROUND: Murraya paniculata (L.) Jack, commonly called orange jessamine in the family Rutaceae, is an important ornamental plant in tropical and subtropical regions which is famous for its strong fragrance. Although genome assemblies have been reported for many Rutaceae species, mainly in the genus Citrus, full genomic information has not been reported for M. paniculata, which is a prerequisite for in-depth genetic studies on Murraya and manipulation using genetic engineering techniques. Here, we report a high-quality chromosome-level genome assembly of M. paniculata and aim to provide insights on the molecular mechanisms of flower volatile biosynthesis. RESULTS: The genome assembly with a contig N50 of 18.25 Mb consists of 9 pseudomolecules and has a total length of 216.86 Mb. Phylogenetic analysis revealed that M. paniculata diverged from the common ancestor approximately 25 million years ago and has not undergone any species-specific whole genome duplication events. Genome structural annotation and comparative genomics analysis revealed that there are obvious differences in transposon contents among the genomes of M. paniculata and Citrus species, especially in the upstream regions of genes. Research on the flower volatiles of M. paniculata and C. maxima at three flowering stages revealed significant differences in volatile composition with the flowers of C. maxima lacking benzaldehyde and phenylacetaldehyde. Notably, there are transposons inserted in the upstream region of the phenylacetaldehyde synthase (PAAS) genes Cg1g029630 and Cg1g029640 in C. maxima, but not in the upstream region of three PAAS genes Me2G_2379, Me2G_2381, and Me2G_2382 in M. paniculata. Our results indicated that compared to the low expression levels of PAAS genes in C. maxima, the higher expression levels of the three PAAS genes in M. paniculata are the main factor affecting the phenylacetaldehyde biosynthesis and causing the content difference of phenylacetaldehyde. The phenylacetaldehyde synthetic activities of the enzymes encoded by M. paniculata PAAS genes were validated by in vitro analyses. CONCLUSIONS: Our study provides useful genomic resources of M. paniculata for further research on Rutaceae plants, identifies new PAAS genes, and provides insights into how transposons contribute to variations in flower volatiles among Murraya and Citrus plants.

Differences in pseudogene evolution contributed to the contrasting flavors of turnip and Chiifu, two Brassica rapa subspecies.

Pseudogenes are important resources for investigation of genome evolution and genomic diversity because they are nonfunctional but have regulatory effects that influence plant adaptation and diversification. However, few systematic comparative analyses of pseudogenes in closely related species have been conducted. Here, we present a turnip (Brassica rapa ssp. rapa) genome sequence and characterize pseudogenes among diploid Brassica species/subspecies. The results revealed that the number of pseudogenes was greatest in Brassica oleracea (CC genome), followed by B. rapa (AA genome) and then Brassica nigra (BB genome), implying that pseudogene differences emerged after species differentiation. In Brassica AA genomes, pseudogenes were distributed asymmetrically on chromosomes because of numerous chromosomal insertions/rearrangements, which contributed to the diversity among subspecies. Pseudogene differences among subspecies were reflected in the flavor-related glucosinolate (GSL) pathway. Specifically, turnip had the highest content of pungent substances, probably because of expansion of the methylthioalkylmalate synthase-encoding gene family in turnips; these genes were converted into pseudogenes in B. rapa ssp. pekinensis (Chiifu). RNA interference-based silencing of the gene encoding 2-oxoglutarate-dependent dioxygenase 2, which is also associated with flavor and anticancer substances in the GSL pathway, resulted in increased abundance of anticancer compounds and decreased pungency of turnip and Chiifu. These findings revealed that pseudogene differences between turnip and Chiifu influenced the evolution of flavor-associated GSL metabolism-related genes, ultimately resulting in the different flavors of turnip and Chiifu.

Effects of tongue hair flexural deformation on viscous fluid transport by bees.

A bee's tongue is coated in dynamic hairs that gradually unfold to entrain the viscid nectar, during which hairs inevitably deflect as a result of fluid drag. The hair deflection induced decline in nectar capture rate may be a coupled elastoviscous problem and remains poorly understood. Here we employed geometric beam theory coupled with the effective viscous force to derive a dynamic model for a rotary tongue hair deflection in a viscous fluid. Considering deflection of the tongue hair, we rationalized the nectar capture rate by takingBombusterrestrisas a model system. When the nectar concentration increases from 20% to 70%, the nectar capture rate declines by 87%, indicating that hair erection is more severely impeded in thicker nectar. Based on this model, we predicted an optimal hair length with which the bee can reach the maximum nectar capture rate. This work may provide a new theoretical framework for quantifying viscous liquid transport by hairy surfaces and shed light on design methodologies for fluid transport devices using hairy beds.

Cold Tolerance of ScCBL6 Is Associated with Tonoplast Transporters and Photosynthesis in Arabidopsis.

Plants that are adapted to harsh environments offer enormous opportunity to understand stress responses in ecological systems. Stipa capillacea is widely distributed in the frigid and arid region of the Tibetan Plateau, but its signal transduction system under cold stress has not been characterized. In this study, we isolated a cDNA encoding the signal transduction protein, ScCBL6, from S. capillacea, and evaluated its role in cold tolerance by ectopically expressing it in Arabidopsis. Full-length ScCBL6 encode 227 amino acids, and are clustered with CBL6 in Stipa purpurea and Oryza sativa in a phylogenetic analysis. Compared with tolerance in wild-type (WT) plants, ScCBL6-overexpressing plants (ScCBL6-OXP) were more tolerant to cold stress but not to drought stress, as confirmed by their high photosynthetic capacity (Fv/Fm) and survival rate under cold stress. We further compared their cold-responsive transcriptome profiles by RNA sequencing. In total, 3931 genes were differentially expressed by the introduction of ScCBL6. These gene products were involved in multiple processes such as the immune system, lipid catabolism, and secondary metabolism. A KEGG pathway analysis revealed that they were mainly enriched in plant hormone signal transduction and biomacromolecule metabolism. Proteins encoded by differentially expressed genes were predicted to be localized in chloroplasts, mitochondria, and vacuoles, suggesting that ScCBL6 exerts a wide range of functions. Based on its tonoplast subcellular location combined with integrated transcriptome and physiological analyses of ScCBL6-OXP, we inferred that ScCBL6 improves plant cold stress tolerance in Arabidopsis via the regulation of photosynthesis, redox status, and tonoplast metabolite transporters.

Optimal kinematics of the bee tongue for viscous fluid transport.

Honey bees can forage nectar from a large spectrum of nectariferous flowers using their rhythmically erectable tongue hairs in a viscous dipping fashion that involves a faster protraction stroke toward the nectar pool and a slower retraction stroke backward. Since honey bees are capable of using their hairy tongues to adapt to various feeding environments, the kinematic characteristics of the bee tongue, especially the retraction time, would likely represent evolutionary optimization. However, the phenomenon and mechanism remain elusive. In this combined experimental and theoretical study, we established a mathematical model to analyze the effects of tongue retraction time on the energy intake rate considering the unfolding dynamics of tongue hairs in the retraction phase. The theoretical optimal retraction time at which the energy intake rate reached the maximum was governed by the dimensions of tongue hairs, which matched well with the in vivo tests. This study may not only bridge the connection between the kinematics and geometry of the bee tongue but also shed light on a control strategy for micropumps equipped with dynamic surfaces.

Establishment of Agrobacterium-mediated genetic transformation and application of CRISPR/Cas9 genome-editing system to Brassica rapa var. rapa.

BACKGROUND: Genome editing is essential for crop molecular breeding. However, gene editing in turnip (Brassica rapa var. rapa) have not been reported owing to the very low transformation efficiency. RESULTS: In this study, we established a transformation procedure involving chemical-inducible activation of the BrrWUSa gene, which resulted in high transformation frequencies of turnip. Estradiol-inducible BrrWUSa transgenic plants were fertile and showed no obvious developmental defects. Furthermore, we used CRISPR/Cas9 gene-editing technology to edit BrrTCP4b and generated 20 BrrTCP4b-edited seedlings with an increase in leaf trichome number. CONCLUSION: The results demonstrate that BrrWUSa improves the regeneration efficiency in turnip. The transformation procedure represents a promising strategy to improve genetic transformation and for functional characterization of genes in turnip.

Hollow mandibles: Structural adaptation to high-speed and powerful strike in the trap-jaw ant Odontomachus monticola.

The trap-jaw ant Odontomachus monticola manipulates its hollow mandibles to generate extremely high speed to impact various objects through a catapult mechanism, making the violent collision occur between the mandible and the impacted objects, which increases the risk of structural failure. However, how the ant balances the trade-off between the powerful clamping and impact resistance by using this hollow structure remains elusive. In this combined experimental and theoretical investigation, we revealed that the hollowness ratio of the mandible plays an essential role in counterbalancing the trade-off. Micro-CT and high-speed images suggested that the hollow mandibles facilitate a high angular acceleration to 108 rad/s2 for an enormous clamping force. However, this hollowness might challenge the structural strength while collision occurs. We found that under the same actuating energy, the von Mises stress of the object collided by the natural mandible striking can reach up to 2.9 times that generated by the entirely solid mandible. We defined the efficiency ratio of the von Mises stress on the impacted object to that on the mandible and found the hollow mandible achieves a more robust balance between powerful clamping and impact resistance compared to the solid mandible.

Quantitative Succinyl-Proteome Profiling of Turnip (Brassica rapa var. rapa) in Response to Cadmium Stress.

Protein post-translational modification (PTM) is an efficient biological mechanism to regulate protein structure and function, but its role in plant responses to heavy metal stress is poorly understood. The present study performed quantitative succinyl-proteome profiling using liquid chromatography−mass spectrometry analysis to explore the potential roles of lysine succinylation modification in turnip seedlings in response to cadmium (Cd) stress (20 µM) under hydroponic conditions over a short time period (0−8 h). A total of 547 succinylated sites on 256 proteins were identified in the shoots of turnip seedlings. These succinylated proteins participated in various biological processes (e.g., photosynthesis, tricarboxylic acid cycle, amino acid metabolism, and response to stimulation) that occurred in diverse cellular compartments according to the functional classification, subcellular localization, and protein interaction network analysis. Quantitative analysis showed that the intensities of nine succinylation sites on eight proteins were significantly altered (p < 0.05) in turnip shoots after 8 h of Cd stress. These differentially succinylated sites were highly conserved in Brassicaceae species and mostly located in the conserved domains of the proteins. Among them, a downregulated succinylation site (K150) in the glycolate oxidase protein (Gene0282600.1), an upregulated succinylation site (K396) in the catalase 3 protein (Gene0163880.1), and a downregulated succinylation site (K197) in the glutathione S-transferase protein (Gene0315380.1) may have contributed to the altered activity of the corresponding enzymes, which suggests that lysine succinylation affects the Cd detoxification process in turnip by regulating the H2O2 accumulation and glutathione metabolism. These results provide novel insights into understanding Cd response mechanisms in plants and important protein modification information for the molecular-assisted breeding of Brassica varieties with distinct Cd tolerance and accumulation capacities.

Inside the coupling of ladybird beetle elytra: elastic setae can facilitate swift deployment.

The ladybird beetle (Coccinella septempunctata) is known for swift deployment of its elytra, an action that requires considerable power. However, actuation by thoracic muscles alone may be insufficient to deploy elytra at high speed because the maximum mechanical power that elytral muscles can produce is only 70% of that required for initiation of deployment. Nevertheless, the elytra open rapidly, within 3 ms in the initial phase, at a maximum angular velocity of 66.49±21.29 rad s-1, rivaling the strike velocity of ant lion (Myrmeleon crudelis) mandibles (65±21 rad s-1). Here, we hypothesize that elytra coupling may function as an energy storage mechanism that facilitates rapid opening by releasing elastic strain energy upon deployment. To test this hypothesis and better understand the biomechanics of elytra deployment, we combined micro-computed tomography and scanning electron microscopy to examine the microstructure of the coupling of paired elytra. We found that two rows of setae on the internal edges of the elytra coupling structure undergo elastic deformation when the elytra are locked together. Kinematics observations and mathematical modeling suggest that the elastic potential energy stored in the compressed setae generates 40% of the power required for deployment of elytra. Our findings broaden insights into how ladybirds actuate elytra opening by a strategy of using both muscles and elastic microstructures, and demonstrate a distributed pattern of actuation that adapts to geometrical constraints in elytra locking.

Genome-Wide Identification and Functional Analysis of the Calcineurin B-like Protein and Calcineurin B-like Protein-Interacting Protein Kinase Gene Families in Chinese Cabbage (Brassica rapa ssp. pekinensis).

In plants, calcineurin B-like proteins (CBL) are a unique set of calcium sensors that decode calcium signals by activating a plant-specific protein kinase family called CBL-interacting protein kinases (CIPKs). The CBL-CIPK family and its interacting complexes regulate plant responses to various environmental stimuli. Chinese cabbage (Brassica rapa ssp. pekinensis) is an important vegetable crop in Asia; however, there are no reports on the role of the CBLs-CIPKs' signaling system in response to abiotic stress during cabbage growth. In this study, 18 CBL genes and 47 CIPK genes were identified from the Chinese cabbage genome. Expansion of the gene families was mainly due to tandem repeats and segmental duplication. An analysis of gene expression patterns showed that different duplicate genes exhibited different expression patterns in response to treatment with Mg2+, K+, and low temperature. In addition, differences in the structural domain sequences of NAF/FISL and interaction profiles in yeast two-hybrid assays suggested a functional divergence of the duplicate genes during the long-term evolution of Chinese cabbage, a result further validated by potassium deficiency treatment using trans-BraCIPK23.1/23.2/23.3 Arabidopsis thaliana. Our results provide a basis for studies related to the functional divergence of duplicate genes and in-depth studies of BraCBL-BraCIPK functions in Chinese cabbage.

Genome-Wide Survey Indicates Diverse Physiological Roles of Dendrobium officinale Calcium-Dependent Protein Kinase Genes.

Calcium-dependent protein kinases (CDPKs) are crucial calcium ions (Ca2+) sensors in plants with important roles in signal transduction, plant growth, development, and stress responses. Here, we identified 24 genes encoding CDPKs in Dendrobium officinale using genome-wide analysis. The phylogenetic analysis revealed that these genes formed four groups, with similar structures in the same group. The gene expression patterns following hormone treatments and yeast two-hybrid of homologous CDPK gene pairs with Rbohs showed differences, indicating functional divergence between homologous genes. In addition, the rapid accumulation of hydrogen peroxide (H2O2) and stomatal closure was observed in response to salicylic acid (SA)/jasmonic acid (JA) stress. Our data showed that CDPK9-2 and CDPK20-4 interacted with Rboh D and Rboh H, respectively, and were implicated in the generation of H2O2 and regulation of the stomatal aperture in response to salicylic acid/jasmonic acid treatment. We believe these results can provide a foundation for the functional divergence of homologous genes in D. officinale.

Functional Characterization of the Stipa purpurea P5CS Gene under Drought Stress Conditions.

Free proline has multiple functions in plant cells, such as regulating osmotic potential and protecting both proteins and cell membranes. The expression of Δ1-Pyrroline-5-carboxylate synthase (P5CS), a key enzyme in the proline biosynthetic pathway, increases under drought, salt and cold stress conditions, causing plant cells to accumulate large amounts of proline. In this study, we cloned and identified the P5CS gene from Stipa purpurea, which has a full-length of 2196 bp and encodes 731 amino acids. A subcellular localization analysis indicated that SpP5CS localized to the cytoplasm. The ectopic overexpression of SpP5CS in Arabidopsis thaliana resulted in higher proline contents, longer roots, higher survival rates and less membrane damage under drought stress conditions compared with wild-type controls. SpP5CS-overexpressing A. thaliana was more resistant to drought stress than the wild type, whereas the deletion mutant sp5cs was less resistant to drought stress. Thus, SpP5CS may be a potential candidate target gene for increasing plant resistance to drought stress.

Effect of calcium ion on the morphology structure and compression elasticity of muscle fibers from honeybee abdomen.

Muscle contraction activated by calcium ion is the key to reveal that honeybee abdomen can achieve various physiological activities through flexible exercises and contributes to a powerful mechanical function of muscle fibers. To investigate the stimulating effect of calcium ion on muscle fibers of honeybee abdomen, atomic force microscopy was used to measure the morphology structure and mechanical properties of muscle fibers from honeybee abdomen in different calcium ion solutions. The periodic morphology structure of muscle fibers stimulated by different calcium ion concentration changed greatly, and the sarcomere length contracted from 6.53 µm to 4.29 µm as the calcium ion concentration increased from 0.11 mM to 10 mM. The mechanical measurement showed that the elastic modulus of Z-line reached the maximum, followed by M-line, overlap zone and I-band in sequence at the same calcium ion concentration, and was approximately 3.636, 2.450, 2.284, 2.748 times that of I-band from 0.11 mM to 10 mM calcium ion concentration. Combining the experimental analysis, the calcium ion threshold range was obtained based on the response surface method. This work adequately elucidates biological structure and biomechanics of muscle fibers from honeybee abdomen and could provide reference for other similar muscle system.

Genome-Wide Identification of OSC Gene Family and Potential Function in the Synthesis of Ursane- and Oleanane-Type Triterpene in Momordica charantia.

The triterpenes in bitter gourd (Momordica charantia) show a variety of medicinal activities. Oxidosqualene cyclase (OSC) plays an indispensable role in the formation of triterpene skeletons during triterpene biosynthesis. In this study, we identified nine genes encoding OSCs from bitter gourd (McOSC1-9). Analyses of their expression patterns in different tissues suggested that characteristic triterpenoids may be biosynthesized in different tissues and then transported. We constructed a hairy root system in which McOSC7 overexpression led to an increased accumulation of camaldulenic acid, enoxolone, and quinovic acid. Thus, the overexpression of McOSC7 increased the active components content in bitter gourd. Our data provide an important foundation for understanding the roles of McOSCs in triterpenoid synthesis.

BrrICE1.1 is associated with putrescine synthesis through regulation of the arginine decarboxylase gene in freezing tolerance of turnip (Brassica rapa var. rapa).

BACKGROUND: In the agricultural areas of Qinghai-Tibet Plateau, temperature varies widely from day to night during the growing season, which makes the extreme temperature become one of the limiting factors of crop yield. Turnip (Brassica rapa var. rapa) is a traditional crop of Tibet grown in the Tibet Plateau, but its molecular and metabolic mechanisms of freezing tolerance are unclear. RESULTS: Here, based on the changes in transcriptional and metabolic levels of Tibetan turnip under freezing treatment, the expression of the arginine decarboxylase gene BrrADC2.2 exhibited an accumulative pattern in accordance with putrescine content. Moreover, we demonstrated that BrrICE1.1 (Inducer of CBF Expression 1) could directly bind to the BrrADC2.2 promoter, activating BrrADC2.2 to promote the accumulation of putrescine, which was verified by RNAi and overexpression analyses for both BrrADC2.2 and BrrICE1.1 using transgenic hair root. The function of putrescine in turnip was further analyzed by exogenous application putrescine and its inhibitor DL-α-(Difluoromethyl) arginine (DFMA) under freezing tolerance. In addition, the BrrICE1.1 was found to be involved in the ICE1-CBF pathway to increase the freezing stress of turnip. CONCLUSIONS: BrrICE1.1 could bind the promoter of BrrADC2.2 or CBFs to participate in freezing tolerance of turnip by transcriptomics and targeted metabolomics analyses. This study revealed the regulatory network of the freezing tolerance process in turnip and increased our understanding of the plateau crops response to extreme environments in Tibet.