Positional cloning and characterization reveal the role of TaSRN-3D and TaBSR1 in the regulation of seminal root number in wheat.

Seminal roots play a critical role in water and nutrient absorption, particularly in the early developmental stages of wheat. However, the genes responsible for controlling SRN in wheat remain largely unknown. Genetic mapping and functional analyses identified a candidate gene (TraesCS3D01G137200, TaSRN-3D) encoding a Ser/Thr kinase glycogen synthase kinase 3 (STKc_GSK3) that regulated SRN in wheat. Additionally, experiments involving hormone treatment, nitrate absorption and protein interaction were conducted to explore the regulatory mechanism of TaSRN-3D. Results showed that the TaSRN-3D4332 allele inhibited seminal roots initiation and development, while loss-of-function mutants showed significantly higher seminal root number (SRN). Exogenous application of epi-brassinolide could increase the SRN in a HS2-allelic background. Furthermore, chlorate sensitivity and 15N uptake assays revealed that a higher number of seminal roots promoted nitrate accumulation. TaBSR1 (BIN2-related SRN Regulator 1, orthologous to OsGRF4/GL2 in rice) acts as an interactor of TaSRN-3D and promotes TaBSR1 degradation to reduce SRN. This study provides valuable insights into understanding the genetic basis and regulatory network of SRN in wheat, highlighting their roles as potential targets for root-based improvement in wheat breeding.

TaNAM-6A is essential for nitrogen remobilisation and regulates grain protein content in wheat (Triticum aestivum L.).

Grain protein content (GPC) is a crucial quality trait in bread wheat, which is influenced by the key transcription factor TaNAM. However, the regulatory mechanisms of TaNAM have remained largely elusive. In this study, a new role of TaNAM was unveiled in regulating nitrogen remobilisation which impacts GPC. The TaNAM knockout mutants generated by clustered regularly interspaced short palindromic repeats/Cas9 exhibited significantly delayed senescence and lower GPC, while overexpression of TaNAM-6A resulted in premature senility and much higher GPC. Further analysis revealed that TaNAM directly activates the genes TaNRT1.1 and TaNPF5.5s, which are involved in nitrogen remobilisation. This activity aids in the transfer of nitrogen from leaves to grains for protein synthesis. In addition, an elite allele of TaNAM-6A, associated with high GPC, was identified as a candidate gene for breeding high-quality wheat. Overall, our work not only elucidates the potential mechanism of TaNAM-6A affecting bread wheat GPC, but also highlights the significance of nitrogen remobilisation from senescent leaves to grains for protein accumulation. Moreover, our research provides a new target and approach for improving the quality traits of wheat, particularly the GPC.

Reshaped DNA methylation cooperating with homoeolog-divergent expression promotes improved root traits in synthesized tetraploid wheat.

Polyploidization is a major event driving plant evolution and domestication. However, how reshaped epigenetic modifications coordinate gene transcription to generate phenotypic variations during wheat polyploidization is currently elusive. Here, we profiled transcriptomes and DNA methylomes of two diploid wheat accessions (SlSl and AA) and their synthetic allotetraploid wheat line (SlSlAA), which displayed elongated root hair and improved root capability for nitrate uptake and assimilation after tetraploidization. Globally decreased DNA methylation levels with a reduced difference between subgenomes were observed in the roots of SlSlAA. DNA methylation changes in first exon showed strong connections with altered transcription during tetraploidization. Homoeolog-specific transcription was associated with biased DNA methylation as shaped by homoeologous sequence variation. The hypomethylated promoters showed significantly enriched binding sites for MYB, which may affect gene transcription in response to root hair growth. Two master regulators in root hair elongation pathway, AlCPC and TuRSL4, exhibited upregulated transcription levels accompanied by hypomethylation in promoter, which may contribute to the elongated root hair. The upregulated nitrate transporter genes, including NPFs and NRTs, also are significantly associated with hypomethylation, indicating an epigenetic-incorporated regulation manner in improving nitrogen use efficiency. Collectively, these results provided new insights into epigenetic changes in response to crop polyploidization and underscored the importance of epigenetic regulation in improving crop traits.

Phase-separated CCER1 coordinates the histone-to-protamine transition and male fertility.

Idiopathic fertility disorders are associated with mutations in various genes. Here, we report that coiled-coil glutamate-rich protein 1 (CCER1), a germline-specific and intrinsically disordered protein (IDP), mediates postmeiotic spermatid differentiation. In contrast, CCER1 deficiency results in defective sperm chromatin compaction and infertility in mice. CCER1 increases transition protein (Tnp1/2) and protamine (Prm1/2) transcription and mediates multiple histone epigenetic modifications during the histone-to-protamine (HTP) transition. Immiscible with heterochromatin in the nucleus, CCER1 self-assembles into a polymer droplet and forms a liquid-liquid phase-separated condensate in the nucleus. Notably, we identified loss-of-function (LoF) variants of human CCER1 (hCCER1) in five patients with nonobstructive azoospermia (NOA) that were absent in 2713 fertile controls. The mutants led to premature termination or frameshift in CCER1 translation, and disrupted condensates in vitro. In conclusion, we propose that nuclear CCER1 is a phase-separated condensate that links histone epigenetic modifications, HTP transitions, chromatin condensation, and male fertility.

Dynamic interactions between E-cadherin and Ankyrin-G mediate epithelial cell polarity maintenance.

E-cadherin is an essential cell‒cell adhesion protein that mediates canonical cadherin-catenin complex formation in epithelial lateral membranes. Ankyrin-G (AnkG), a scaffold protein linking membrane proteins to the spectrin-based cytoskeleton, coordinates with E-cadherin to maintain epithelial cell polarity. However, the molecular mechanisms governing this complex formation and its relationships with the cadherin-catenin complex remain elusive. Here, we report that AnkG employs a promiscuous manner to encapsulate three discrete sites of E-cadherin by the same region, a dynamic mechanism that is distinct from the canonical 1:1 molar ratio previously described for other AnkG or E-cadherin-mediated complexes. Moreover, we demonstrate that AnkG-binding-deficient E-cadherin exhibited defective accumulation at the lateral membranes and show that disruption of interactions resulted in cell polarity malfunction. Finally, we demonstrate that E-cadherin is capable of simultaneously anchoring to AnkG and ß-catenin, providing mechanistic insights into the functional orchestration of the ankyrin-spectrin complex with the cadherin-catenin complex. Collectively, our results show that complex formation between E-cadherin and AnkG is dynamic, which enables the maintenance of epithelial cell polarity by ensuring faithful targeting of the adhesion molecule-scaffold protein complex, thus providing molecular mechanisms for essential E-cadherin-mediated complex assembly at cell‒cell junctions.

The TaTCP4/10-B1 cascade regulates awn elongation in wheat (Triticum aestivum L.).

Awns are important morphological markers for wheat and exert a strong physiological effect on wheat yield. The awn elongation suppressor B1 has recently been cloned through association and linkage analysis in wheat. However, the mechanism of awn inhibition centered around B1 remains to be clarified. Here, we identified an allelic variant in the coding region of B1 through analysis of re-sequencing data; this variant causes an amino acid substitution and premature termination, resulting in a long-awn phenotype. Transcriptome analysis indicated that B1 inhibited awn elongation by impeding cytokinin- and auxin-promoted cell division. Moreover, B1 directly repressed the expression of TaRAE2 and TaLks2, whose orthologs have been reported to promote awn development in rice or barley. More importantly, we found that TaTCP4 and TaTCP10 synergistically inhibited the expression of B1, and a G-to-A mutation in the B1 promoter attenuated its inhibition by TaTCP4/10. Taken together, our results reveal novel mechanisms of awn development and provide genetic resources for trait improvement in wheat.

Associations between mercury exposure with blood pressure and lipid levels: A cross-sectional study of dental professionals.

Mercury (Hg) exposure is a public health problem worldwide that is now being addressed through the Minamata Convention on Mercury. Fish containing methylmercury and dental amalgam containing elemental Hg are the major sources of exposure for most populations. There is some evidence that methylmercury impacts cardiovascular and metabolic health, primarily in populations with high exposure levels. Studies of elemental Hg and these outcomes are relatively rare. We aimed to examine associations between Hg exposure (both elemental and methylmercury) and blood pressure, as well as cholesterol and triglyceride levels. In 2012, we recruited dental professionals attending the Health Screening Program at the American Dental Association (ADA) Annual Session in California. Total Hg levels in hair and blood samples were analyzed as indicators of methylmercury exposure and in urine as an indicator of primarily elemental Hg exposure (n = 386; mean ± sd age 55 ± 11 years). We measured blood pressure (systolic and diastolic) and lipid profiles (total cholesterol, high-density lipoprotein cholesterol [HDL], low-density lipoprotein cholesterol [LDL] and triglycerides). The geometric means (geometric standard deviations) for blood, hair, and urine Hg were 3.64 (2.39) µg/L, 0.60 (2.91) µg/g, and 1.30 (2.44) µg/L, respectively. For every one µg/L increase in specific gravity-adjusted urine Hg, LDL increased by 2.31 mg/dL (95% CI = 0.09, 4.54), in linear regression adjusting for BMI, race, sex, polyunsaturated fatty acid intake from fish consumption, smoking status, and use of cholesterol-lowering medication. No significant associations between Hg biomarkers and blood pressure or hair or blood Hg with lipid levels were observed. Results suggest that elemental Hg exposure may influence LDL concentrations in adults with low-level exposure, and this relationship merits further study in other populations.

Genome-Wide Association Analysis Reveals the Genetic Basis of Iron-Deficiency Stress Tolerance in Maize.

Iron (Fe) is an essential trace element for almost all organisms and is often the major limiting nutrient for normal growth. Fe deficiency is a worldwide agricultural problem, which affects crop productivity and product quality. Understanding the Fe-deficiency response in plants is necessary for improving both plant health and the human diet. In this study, Fe-efficient (Ye478) and Fe-inefficient maize inbred lines (Wu312) were used to identify the genotypic difference in response to low Fe stress during different developmental stages and to further determine the optimal Fe-deficient Fe(II) supply level which leads to the largest phenotypic difference between Ye478 and Wu312. Then, genome-wide association analysis was performed to further identify candidate genes associated with the molecular mechanisms under different Fe nutritional statuses. Three candidate genes involved in Fe homeostasis of strategy II plants (strategy II genes) were identified, including ZmDMAS1, ZmNAAT1, and ZmYSL11. Furthermore, candidate genes ZmNAAT1, ZmDMAS1, and ZmYSL11 were induced in Fe-deficient roots and shoots, and the expression of ZmNAAT1 and ZmDMAS1 responded to Fe deficiency more in shoots than in roots. Beyond that, several genes that may participate in Fe homeostasis of strategy I plants (strategy I genes) were identified, which were either encoding Fe transporters (ZmIRT1 and ZmZIP4), or acting as essential ethylene signal transducers (ZmEBF1). Interestingly, ZmIRT1, ZmZIP4, and ZmEBF1 were significantly upregulated under low Fe stress, suggesting that these genes may be involved in Fe-deficiency tolerance in maize which is considered as strategy II plant. This study demonstrates the use of natural variation in the association population to identify important genes associated with Fe-deficiency tolerance and may further provide insights for understanding the molecular mechanism underlying the tolerance to Fe-deficiency stress in maize.

Transcription factor-like 5 is a potential DNA- and RNA-binding protein essential for maintaining male fertility in mice.

Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, but the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion as the heterozygous males (Tcfl5+/-) were infertile. However, we did observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally and post-transcriptionally regulated a set of genes participating in male germ cell development via TCFL5 ChIP-DNA and eCLIP-RNA high-throughput sequencing. We also identified a known RNA-binding protein, FXR1, as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and acts as a dual-function protein that mediates DNA and RNA to regulate spermatogenesis. This article has an associated First Person interview with the first author of the paper.

Assembly status transition offers an avenue for activity modulation of a supramolecular enzyme.

Nature has evolved many supramolecular proteins assembled in certain, sometimes even seemingly oversophisticated, morphological manners. The rationale behind such evolutionary efforts is often poorly understood. Here, we provide atomic-resolution insights into how the dynamic building of a structurally complex enzyme with higher order symmetry offers amenability to intricate regulation. We have established the functional coupling between enzymatic activity and protein morphological states of glutamine synthetase (GS), an old multi-subunit enzyme essential for cellular nitrogen metabolism. Cryo-EM structure determination of GS in both the catalytically active and inactive assembly states allows us to reveal an unanticipated self-assembly-induced disorder-order transition paradigm, in which the remote interactions between two subcomplex entities significantly rigidify the otherwise structurally fluctuating active sites, thereby regulating activity. We further show in vivo evidences that how the enzyme morphology transitions could be modulated by cellular factors on demand. Collectively, our data present an example of how assembly status transition offers an avenue for activity modulation, and sharpens our mechanistic understanding of the complex functional and regulatory properties of supramolecular enzymes.

Arabidopsis G-Protein ß Subunit AGB1 Negatively Regulates DNA Binding of MYB62, a Suppressor in the Gibberellin Pathway.

Plant G proteins are versatile components of transmembrane signaling transduction pathways. The deficient mutant of heterotrimeric G protein leads to defects in plant growth and development, suggesting that it regulates the GA pathway in Arabidopsis. However, the molecular mechanism of G protein regulation of the GA pathway is not understood in plants. In this study, two G protein ß subunit (AGB1) mutants, agb1-2 and N692967, were dwarfed after exogenous application of GA3. AGB1 interacts with the DNA-binding domain MYB62, a GA pathway suppressor. Transgenic plants were obtained through overexpression of MYB62 in two backgrounds including the wild-type (MYB62/WT Col-0) and agb1 mutants (MYB62/agb1) in Arabidopsis. Genetic analysis showed that under GA3 treatment, the height of the transgenic plants MYB62/WT and MYB62/agb1 was lower than that of WT. The height of MYB62/agb1 plants was closer to MYB62/WT plants and higher than that of mutants agb1-2 and N692967, suggesting that MYB62 is downstream of AGB1 in the GA pathway. qRT-PCR and competitive DNA binding assays indicated that MYB62 can bind MYB elements in the promoter of GA2ox7, a GA degradation gene, to activate GA2ox7 transcription. AGB1 affected binding of MYB62 on the promoter of GA2ox7, thereby negatively regulating th eactivity of MYB62.

Sandwiched-fusion strategy facilitates recombinant production of small labile proteins.

Efficient production of large quantities of soluble, properly folded proteins is of high demand in modern structural and functional genomics. Despite much advancement toward improving recombinant protein expression, many eukaryotic proteins especially small peptides often fail to be recovered due to rapid proteolytic degradation. Here we show that the sandwiched-fusion strategy, which is based on two protein tags incorporated both at the amino- and carboxyl-terminus of target protein, could be employed to overcome this obstacle. We have exploited this strategy on heterologous expression in Escherichia coli of eight small degradation-prone eukaryotic proteins, whose successful recombinant productions have yet to be achieved. These include seven mitochondria-derived peptides (MDPS), a class of unique metabolic regulators of human body, and a labile mosquito transcription factor, Guy1. We show here that the sandwiched-fusion strategy, which provides robust protection against proteolysis, affords an economical method to obtain large quantities of pure five MDPs and the transcription factor Guy1, in sharp contrast to otherwise unsuccessful recovery using the traditional amino-fusion method. Further biophysical characterization and interaction studies by NMR spectroscopy confirmed that the proteins produced by this novel approach are properly folded into their biologically active structures. We anticipate this strategy could be widely utilized in production of other labile protein systems.

SiMYB56 Confers Drought Stress Tolerance in Transgenic Rice by Regulating Lignin Biosynthesis and ABA Signaling Pathway.

Foxtail millet (Setaria italica) originated in China and is generally cultivated in arid and barren soil. Through long-term harsh environmental selection, foxtail millet has acquired significant drought resistance. However, the molecular mechanism of foxtail millet drought resistance is still unknown. Here, we identified a drought-induced R2R3-MYB transcription factor SiMYB56 in foxtail millet. Overexpression of SiMYB56 significantly enhances tolerance to drought stress in transgenic rice plants at both the vegetative and the reproductive stage and has no adverse effect on its normal growth. Compared with wild-type controls, SiMYB56-overexpressing rice plants had lower MDA content and higher lignin content under drought conditions. Quantitative real-time PCR and Transcriptional activity assays demonstrated that SiMYB56 could activate expression of lignin biosynthesis genes under drought conditions. Also, we found that overexpression of SiMYB56 can led to ABA accumulation in the seeds transgenic rice plants. Further experiments showed that Overexpression of SiMYB56 can upregulate the expression of ABA synthesis and response related genes under drought conditions. In conclusion, SiMYB56 may enhance the drought resistance of transgenic rice plants by regulating lignin biosynthesis and ABA signaling pathway, making SiMYB56 a candidate gene for drought resistance improvement in gramineous crops.

mRBPome capture identifies the RNA-binding protein TRIM71, an essential regulator of spermatogonial differentiation.

Continual spermatogenesis relies on the actions of an undifferentiated spermatogonial population that is composed of stem cells and progenitors. Here, using mouse models, we explored the role of RNA-binding proteins (RBPs) in regulation of the biological activities of this population. Proteins bound to polyadenylated RNAs in primary cultures of undifferentiated spermatogonia were captured with oligo (dT)-conjugated beads after UV-crosslinking and profiled by proteomics (termed mRBPome capture), yielding a putative repertoire of 473 RBPs. From this database, the RBP TRIM71 was identified and found to be expressed by stem and progenitor spermatogonia in prepubertal and adult mouse testes. Tissue-specific deletion of TRIM71 in the male germline led to reduction of the undifferentiated spermatogonial population and a block in transition to the differentiating state. Collectively, these findings demonstrate a key role of the RBP system in regulation of the spermatogenic lineage and may provide clues about the influence of RBPs on the biology of progenitor cell populations in other lineages.

Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus.

Animals exploit different germ-line-encoded proteins with various domain structures to detect the signature molecules of pathogenic microbes. These molecules are known as pathogen-associated molecular patterns (PAMPs), and the host proteins that react with PAMPs are called pattern recognition proteins (PRPs). Here, we present a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). This domain is designated as apextrin C-terminal domain (ApeC), and its presence was confirmed in several invertebrate phyla and subphyla. Two apextrin-like proteins (ALP1 and ALP2) were identified in a basal chordate, the Japanese amphioxus Branchiostoma japonicum (bj). bjALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram-positive bacterium Staphylococcus aureus via PGN binding. Neutralization of secreted bjALP1 by anti-bjALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. bjALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB pathway. BjALP1 and bjALP2 therefore play distinct and complementary functions in amphioxus gut mucosal immunity. In conclusion, discovery of the ApeC domain and the functional analyses of amphioxus ALP1 and ALP2 allowed us to define a previously undocumented type of PRP that is represented across different animal phyla.

Experimental investigation on consistency limits of cement and lime-stabilized marine sediments.

This paper presents the effects of treatments with cement and lime on the consistency limits of marine sediments dredged from Dunkirk port. The Casagrande percussion test and the fall cone test were used to determine the liquid limits of raw sediments and treated marine sediments. For the evaluation of the plastic limits, the results of the fall cone test were compared with those obtained by the rolling test method. The relationship between the water contents and the penetration depths for the determination of the liquid limit and the plastic limit was explored. Liquid limits at 15.5 mm and plastic limits at 1.55 mm seem to be a more appropriate choice for the studied marine sediments compared with the limits determined by other used prediction methods. Finally, the effect of cement treatment and lime treatment on the Casagrande classification of the studied sediments was investigated according to the different prediction results.

A closer look at the spectroscopic properties of possible reaction intermediates in wild-type and mutant (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH or LytB) catalyzes the terminal step of the MEP/DOXP pathway where it converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into the two products, isopentenyl diphosphate and dimethylallyl diphosphate. The reaction involves the reductive elimination of the C4 hydroxyl group, using a total of two electrons. Here we show that the active form of IspH contains a [4Fe-4S] cluster and not the [3Fe-4S] form. Our studies show that the cluster is the direct electron source for the reaction and that a reaction intermediate is bound directly to the cluster. This active form has been trapped in a state, dubbed FeS(A), that was detected by electron paramagnetic resonance (EPR) spectroscopy when one-electron-reduced IspH was incubated with HMBPP. In addition, three mutants of IspH have been prepared and studied, His42, His124, and Glu126 (Aquifex aeolicus numbering), with particular attention paid to the effects on the cluster properties and possible reaction intermediates. None of the mutants significantly affected the properties of the [4Fe-4S](+) cluster, but different effects were observed when one-electron-reduced forms were incubated with HMBPP. Replacing His42 led to an increased K(M) value and a much lower catalytic efficiency, confirming the role of this residue in substrate binding. Replacing the His124 also resulted in a lower catalytic efficiency. In this case, however, the enzyme showed the loss of the [4Fe-4S](+) EPR signal upon addition of HMBPP without the subsequent formation of the FeS(A) signal. Instead, a radical-type signal was observed in some of the samples, indicating that this residue plays a role in the correct positioning of the substrate. The incorrect orientation in the mutant leads to the formation of substrate-based radicals instead of the cluster-bound intermediate complex FeS(A). Replacing the Glu126 also resulted in a lower catalytic efficiency, with yet a third type of EPR signal being detected upon incubation with HMBPP. (31)P and (2)H ENDOR measurements of the FeS(A) species incubated with regular and (2)H-C4-labeled HMBPP reveal that the substrate binds to the enzyme in the proximity of the active-site cluster with C4 adjacent to the site of linkage between the FeS cluster and HMBPP. Comparison of the spectroscopic properties of this intermediate to those of intermediates detected in (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase and ferredoxin:thioredoxin reductase suggests that HMBPP binds to the FeS cluster via its hydroxyl group instead of a side-on binding as previously proposed for the species detected in the inactive Glu126 variant. Consequences for the IspH reaction mechanism are discussed.

Solidification/stabilization of dredged marine sediments for road construction.

Cement/lime-based solidification is an environmentally sound solution for the management of dredged marine sediments, instead of traditional solutions such as immersion. Based on the mineralogical composition and physical characteristics of Dunkirk sediments, the effects of cement and lime are assessed through Atterberg limits, modified Proctor compaction, unconfined compressive strength and indirect tensile strength tests. The variation of Atterberg limits and the improvement in strength are discussed at different binder contents. The potential of sediments solidified with cement or lime for road construction is evaluated through a proposed methodology from two aspects: I-CBR value and material classification. The test results show the feasibility of solidified dredged sediments for beneficial use as a material in road construction. Cement is superior to lime in terms of strength improvement, and adding 6% cement is an economic and reasonable method to stabilize fine sediments.