Dynamic changes of rumen bacteria and their fermentative ability in high-producing dairy cows during the late perinatal period.

Background: High-producing dairy cows face varying degrees of metabolic stress and challenges during the late perinatal period, resulting in ruminal bacteria abundance and their fermentative ability occurring as a series of changes. However, the dynamic changes are still not clear. Aims/methods: Ten healthy, high-producing Holstein dairy cows with similar body conditions and the same parity were selected, and ruminal fluid from the dairy cows at postpartum 0, 7, 14, and 21 d was collected before morning feeding. 16S rRNA high-throughput sequencing, GC-MS/MS targeted metabolomics, and UPLC-MS/MS untargeted metabolomics were applied in the study to investigate the dynamic changes within 21 d postpartum. Results: The results displayed that the structures of ruminal bacteria were significantly altered from 0 to 7 d postpartum (R = 0.486, P = 0.002), reflecting the significantly declining abundances of Euryarchaeota and Chloroflexi phyla and Christensenellaceae, Methanobrevibacter, and Flexilinea genera (P < 0.05) and the obviously ascending abundances of Ruminococcaceae, Moryella, Pseudobutyrivibrio, and Prevotellaceae genera at 7 d postpartum (P < 0.05). The structures of ruminal bacteria also varied significantly from 7 to 14 d postpartum (R = 0.125, P = 0.022), reflecting the reducing abundances of Christensenellaceae, Ruminococcaceae, and Moryella genera (P < 0.05), and the elevating abundances of Sharpea and Olsenella genera at 14 d postpartum (P < 0.05). The metabolic profiles of ruminal SCFAs were obviously varied from 0 to 7 d postpartum, resulting in higher levels of propionic acid, butyric acid, and valeric acid at 7 d postpartum (P < 0.05); the metabolic profiles of other ruminal metabolites were significantly shifted from 0 to 7 d postpartum, with 27 significantly elevated metabolites and 35 apparently reduced metabolites (P < 0.05). The correlation analysis indicated that propionic acid was positively correlated with Prevotellaceae and Ruminococcaceae (P < 0.05), negatively correlated with Methanobrevibacter (P < 0.01); butyric acid was positively associated with Prevotellaceae, Ruminococcaceae, and Pseudobutyrivibrio (P < 0.05), negatively associated with Christensenellaceae (P < 0.01); valeric acid was positively linked with Prevotellaceae and Ruminococcaceae (P < 0.05); pyridoxal was positively correlated with Flexilinea and Methanobrevibacter (P < 0.05) and negatively correlated with Ruminococcaceae (P < 0.01); tyramine was negatively linked with Ruminococcaceae (P < 0.01). Conclusion: The findings contribute to the decision of nutritional management and prevention of metabolic diseases in high-producing dairy cows during the late perinatal period.

Genome-Wide Analysis of the Wall-Associated Kinase (WAK) Genes in Medicago truncatula and Functional Characterization of MtWAK24 in Response to Pathogen Infection.

The wall-associated kinases (WAKs) can perceive and transmit extracellular signals as one kind of unique receptor-like kinases (RLKs) involved in the regulation of cell expansion, pathogen resistance and abiotic stress tolerance. To understand their potential roles and screen some key candidates in Medicago truncatula (M. truncatula), genome-wide identification and characterization of MtWAKs were conducted in this study. A total of 54 MtWAK genes were identified and classified into four groups based on their protein domains. They were distributed on all chromosomes, while most of them were clustered on chromosome 1 and 3. The synteny analysis showed that 11 orthologous pairs were identified between M. truncatula and Arabidopsis thaliana (A. thaliana) and 31 pairs between M. truncatula and Glycine max (G. max). The phylogenetic analysis showed that WAK-RLKs were classified into five clades, and they exhibited a species-specific expansion. Most MtWAK-RLKs had similar exon-intron organization and motif distribution. Multiple cis-acting elements responsive to phytohormones, stresses, growth and development were observed in the promoter regions of MtWAK-RLKs. In addition, the expression patterns of MtWAK-RLKs varied with different plant tissues, developmental stages and biotic and abiotic stresses. Interestingly, plasm membrane localized MtWAK24 significantly inhibited Phytophthora infection in tobacco. The study provides valuable information for characterizing the molecular functions of MtWAKs in regulation of plant growth, development and stress tolerance in legume plants.

Influence of Parturition on Rumen Bacteria and SCFAs in Holstein Cows Based on 16S rRNA Sequencing and Targeted Metabolomics.

The rumen fluids from ten cows at Day 3~5 before calving and Day 0 after calving were collected to analyze the composition and quantity of bacterial communities and concentrations of SCFAs. The results showed that the relative abundances of unidentified Lachnospiraceae, Acetitomaculum, Methanobrevibacter, Olsenella, Syntrophococcus, Lachnospira, and Lactobacillus genera were significant increased (p < 0.05), while that of unidentified-Prevotellaceae was notably decreased after calving (p < 0.05). In addition, the concentrations of acetic acid, propionic acid, butyric acid, and caproic acid obviously decreased after calving (p < 0.01). Our findings show that parturition altered the rumen microbiota and their fermentation ability in dairy cows. This study defines a rumen bacteria and metabolic profile of SCFAs associated with parturition in dairy cows.

3D face recognition algorithm based on deep Laplacian pyramid under the normalization of epidemic control.

Under the normalization of epidemic control in COVID-19, it is essential to realize fast and high-precision face recognition without feeling for epidemic prevention and control. This paper proposes an innovative Laplacian pyramid algorithm for deep 3D face recognition, which can be used in public. Through multi-mode fusion, dense 3D alignment and multi-scale residual fusion are ensured. Firstly, the 2D to 3D structure representation method is used to fully correlate the information of crucial points, and dense alignment modeling is carried out. Then, based on the 3D critical point model, a five-layer Laplacian depth network is constructed. High-precision recognition can be achieved by multi-scale and multi-modal mapping and reconstruction of 3D face depth images. Finally, in the training process, the multi-scale residual weight is embedded into the loss function to improve the network's performance. In addition, to achieve high real-time performance, our network is designed in an end-to-end cascade. While ensuring the accuracy of identification, it guarantees personnel screening under the normalization of epidemic control. This ensures fast and high-precision face recognition and establishes a 3D face database. This method is adaptable and robust in harsh, low light, and noise environments. Moreover, it can complete face reconstruction and recognize various skin colors and postures.

An appropriate ammonium: nitrate ratio promotes the growth of centipedegrass: insight from physiological and micromorphological analyses.

Reasonable nitrogen fertilizer application is an important strategy to maintain optimal growth of grasslands, thereby enabling them to better fulfil their ecological functions while reducing environmental pollution caused by high nitrogen fertilizer production and application. Optimizing the ammonium (NH4 +):nitrate (NO3 -) ratio is a common approach for growth promotion in crops and vegetables, but research on this topic in grass plants has not received sufficient attention. Centipedegrass, which is widely used in landscaping and ecological protection, was used as the experimental material. Different NH4 +:NO3 - ratios (0: 100, 25:75, 50:50, 75:25, 100:0) were used as the experimental treatments under hydroponic conditions. By monitoring the physiological and morphological changes under each treatment, the appropriate NH4 +:NO3 - ratio for growth and its underlying mechanism were determined. As the proportion of ammonium increased, the growth showed a "bell-shaped" response, with the maximum biomass and total carbon and nitrogen accumulation achieved with the NH4 +:NO3 - ratio of 50:50 treatment. Compared with the situation where nitrate was supplied alone, increasing the ammonium proportion increased the whole plant biomass by 93.2%, 139.7%, 59.0%, and 30.5%, the whole plant nitrogen accumulation by 44.9%, 94.6%, 32.8%, and 54.8%, and the whole plant carbon accumulation by 90.4%, 139.9%, 58.7%, and 26.6% in order. As a gateway for nitrogen input, the roots treated with an NH4 +:NO3 - ratio of 50:50 exhibited the highest ammonium and nitrate uptake rate, which may be related to the maximum total root length, root surface area, average root diameter, root volume, and largest root xylem vessel. As a gateway for carbon input, leaves treated with an NH4 +:NO3 - ratio of 50:50 exhibited the highest stomatal aperture, stomatal conductance, photosynthetic rate, transpiration rate, and photosynthetic products. The NH4 +:NO3 - ratio of 50:50 treatment had the largest stem xylem vessel area. This structure and force caused by transpiration may synergistically facilitate root-to-shoot nutrient translocation. Notably, the change in stomatal opening occurred in the early stage (4 hours) of the NH4 +:NO3 - ratio treatments, indicating that stomates are structures that are involved in the response to changes in the root NH4 +:NO3 - ratio. In summary, we recommend 50:50 as the appropriate NH4 +:NO3 - ratio for the growth of centipedegrass, which not only improves the nitrogen use efficiency but also enhances the carbon sequestration capacity.

Mining for salt-tolerant genes from halophyte Zoysia matrella using FOX system and functional analysis of ZmGnTL.

Zoysia matrella is a salt-tolerant turfgrass grown in areas with high soil salinity irrigated with effluent water. Previous studies focused on explaining the regulatory mechanism of Z. matrella salt-tolerance at phenotypic and physiological levels. However, the molecular mechanism associated with salt tolerance of Z. matrella remained unclear. In this study, a high-efficient method named FOX (full-length cDNA overexpression) hunting system was used to search for salt-tolerant genes in Z. matrella. Eleven candidate genes, including several known or novel salt-tolerant genes involved in different metabolism pathways, were identified. These genes exhibited inducible expression under salt stress condition. Furthermore, a novel salt-inducible candidate gene ZmGnTL was transformed into Arabidopsis for functional analysis. ZmGnTL improved salt-tolerance through regulating ion homeostasis, reactive oxygen species scavenging, and osmotic adjustment. In summary, we demonstrated that FOX is a reliable system for discovering novel genes relevant to salt tolerance and several candidate genes were identified from Z. matrella that can assist molecular breeding for plant salt-tolerance improvement.

Using an ultra-compact optical system to improve lateral flow immunoassay results quantitatively.

The lateral flow immunoassay (LFIA) is a paper-based platform with extensive application in point-of-care (POC) testing and many fields. However, its clinical application is severely limited due to the lack of quantitative ability of standard LFIA tests; this augmentation provides the system with quantifying the signal from magenta-colored AuNPs. To address this issue, we proposed an ultra-compact optical system that allowed LFIAs to be performed more accurately and objectively. The experimental setup consisted of multiple optical accessories manufactured by 3D printing (STEP files were included). A high-resolution printer was used to print out a magenta card model for the LFIA, whose color code, ranging from 255, 255, 255 to 255, 0, 255 in the RGB (red, green, blue) format, represents different levels of magenta color intensity (from 0% to 100%) and thus the results of LFIA test strips. A mathematical model was built using a calibration curve to describe the relationship between magenta color value and reflectance spectrum. In addition, a spectrum module was integrated into the proposed system to identify and quantify LFIA results. This integration represents a pioneering step in developing portable detection techniques that facilitate quantifying LFIA results. Finally, we expect this ultra-compact optical spectroscopy system to have great potential for novel clinical applications.

A Faster, Novel Technique to Detect COVID-19 Neutralizing Antibodies.

BACKGROUND The COVID-19 pandemic has spread globally in a short period of time. It is known that antibody (nAb) level can effectively predict vaccine efficacy, which leads to the exploration of vaccine trials for efficacy assessment. Thus, the current study aimed to develop a platform to quantify nAb levels faster, at lower cost, and with better efficiency. MATERIAL AND METHODS A total of 69 sera samples were collected for the research, 28 of which were from unvaccinated participants. The other 27 samples and the remaining 14 samples were from the participants who had received the first and second dose, respectively, of AZ vaccine 1 month before. With cPass assays (Genscript cPass nAb ELISA assay) used as a criterion standard and lateral flow immunoassay kit (Healgen Scientific - LFIA test kit) coupled with a spectrometer (LFIA+S) for checking each specimen, we aimed to detect the presence of neutralizing antibodies in sera and to confirm the relationship between the inhibition rate from cPass assays and the nAb index from the LFIA+S. RESULTS Data analysis of the research were taken from the certified ELISA and LFIA+S, which indicated a high consistency (Pearson's r =0.864; ICC=0.90138) between the 2 methods. CONCLUSIONS The dataset demonstrated that LFIA+S was affordable, had a strong correlation with results of the cPass nAbs detection kit, and has potential clinical applications, with an exclusive feature that allows non-experts to use it with ease. It is believed that the proposed platform can be promoted in the near future.

Heterodimer formed by ROC8 and ROC5 modulates leaf rolling in rice.

Moderately rolled leaf is one of the target traits of the ideal plant architecture in rice breeding. Many genes, including homeodomain leucine zipper IV transcription factors ROC5 and ROC8, regulating rice leaf rolling have been cloned and functionally analysed. However, the molecular mechanism by which these genes modulate leaf-rolling remains largely elusive. In this study, we demonstrated the transcription activation activity of both ROC8 and ROC5. Overexpressing ROC8 caused adaxially rolled leaves due to decreased number and size of bulliform cells, whereas knockout of ROC8 induced abaxially rolled leaves due to increased number and size of bulliform cells. ROC8 and ROC5 each could form homodimer, but ROC8 interacted preferably with ROC5 to forms a heterodimer. Importantly, we showed that the ROC8-ROC5 heterodimer rather than the homodimer of ROC8 or ROC5 was functional as neither overexpressing ROC8 in the ROC5 mutant nor overexpressing ROC5 in the ROC8-knockout line could rescue the mutant phenotype. This was further partially supported by the identification of a large number of common differentially expressed genes in single and double mutants of roc8 and roc5. ROC8 and ROC5 were functionally additive as the phenotype of abaxially rolled leaves was stronger in the roc5roc8 double mutant than in their single mutants. Our results provide evidence for the role of dimerization of ROC members in regulating leaf rolling of rice.

Genome-Wide Identification and Characterization of Calcium-Dependent Protein Kinase (CDPK) and CDPK-Related Kinase (CRK) Gene Families in Medicago truncatula.

Calcium-dependent protein kinase (CDPK or CPK) and CDPK-related kinase (CRK) play an important role in plant growth, development, and adaptation to environmental stresses. However, their gene families had been yet inadequately investigated in Medicago truncatula. In this study, six MtCRK genes were computationally identified, they were classified into five groups with MtCDPKs based on phylogenetic relationships. Six pairs of segmental duplications were observed in MtCDPK and MtCRK genes and the Ka/Ks ratio, an indicator of selection pressure, was below 0.310, indicating that these gene pairs underwent strong purifying selection. Cis-acting elements of morphogenesis, multiple hormone responses, and abiotic stresses were predicted in the promoter region. The spatial expression of MtCDPKs and MtCRKs displays diversity. The expression of MtCDPKs and MtCRKs could be regulated by various stresses. MtCDPK4, 14, 16, 22, and MtCRK6 harbor both N-myristoylation site and palmitoylation site and were anchored on plasma membrane, while MtCDPK7, 9, and 15 contain no or only one N-acylation site and were distributed in cytosol and nucleus, suggesting that the N-terminal acylation sites play a key role in subcellular localization of MtCDPKs and MtCRKs. In summary, comprehensive characterization of MtCDPKs and MtCRKs provide a subset of candidate genes for further functional analysis and genetic improvement against drought, cold, salt and biotic stress.

Earlier Degraded Tapetum1 (EDT1) Encodes an ATP-Citrate Lyase Required for Tapetum Programmed Cell Death.

In flowering plants, the tapetum cells in anthers undergo programmed cell death (PCD) at the late meiotic stage, providing nutrients for further development of microspores, including the formation of the pollen wall. However, the molecular basis of tapetum PCD remains elusive. Here we report a tapetum PCD-related mutant in rice (Oryza sativa), earlier degraded tapetum 1 (edt1), that shows complete pollen abortion associated with earlier-than-programmed tapetum cell death. EDT1 encodes a subunit of ATP-citrate lyase (ACL), and is specifically expressed in the tapetum of anthers. EDT1 localized in both the nucleus and the cytoplasm as observed in rice protoplast transient assays. We demonstrated that the A and B subunits of ACL interacted with each other and might function as a heteromultimer in the cytoplasm. EDT1 catalyzes the critical steps in cytosolic acetyl-CoA synthesis. Our data indicated a decrease in ATP level, energy charge, and fatty acid content in mutant edt1 anthers. In addition, the genes encoding secretory proteases or lipid transporters, and the transcription factors known to regulate PCD, were downregulated. Our results demonstrate that the timing of tapetum PCD must be tightly regulated for successful pollen development, and that EDT1 is involved in the tapetum PCD process. This study furthers our understanding of the molecular basis of pollen fertility and fecundity in rice and may also be relevant to other flowering plants.

A GARP transcription factor anther dehiscence defected 1 (OsADD1) regulates rice anther dehiscence.

Anther dehiscence, one of the essential steps in pollination and double fertilization, is regulated by a complex signaling pathway encompassing hormones and environmental factors. However, key components underlying the signaling pathway that regulate anther dehiscence remain largely elusive. Here, we isolated a rice mutant anther dehiscence defected 1 (Osadd1) that exhibited defects in anther dehiscence and glume open. Map-based cloning revealed that OsADD1 encoded a GARP (Golden2, ARR-B and Psr1) transcription factor. Sequence analysis showed that a single base deletion in Osadd1 mutant resulted in pre-termination of the GARP domain. OsADD1 was constitutively expressed in various tissues, with more abundance in the panicles. The major genes associated with anther dehiscence were affected in the Osadd1 mutant, and the expression level of the cellulose synthase-like D sub-family 4 (OsCSLD4) was significantly decreased. We demonstrate that OsADD1 regulated the expression of OsCSLD4 by binding to its promoter, and affects rice anther dehiscence.

Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast development and establishment of the plastidic ribosome system in rice.

The chloroplast is an important organelle that performs photosynthesis as well as biosynthesis and storage of many metabolites. Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in protein synthesis. However, the relationship between chloroplast development and aaRSs still remains unclear. In this study, we isolated a rice albino 1 (ra1) mutant through methane sulfonate (EMS) mutagenesis of rice japonica cultivar Ningjing 4 (Oryza sativa L.), which displayed albinic leaves in seedling stage due to abnormal chloroplast development. Compared with wild type (WT), ra1 showed significantly decreased levels of chlorophylls (Chl) and carotenoids (Car) in 2-week-old seedlings, which also showed obvious plastidic structural defects including abnormal thylakoid membrane structures and more osmiophilic particles. These defects caused albino phenotypes in seedlings. Map-based cloning revealed that RA1 gene encodes a glycyl-tRNA synthetase (GlyRS), which was confirmed by genetic complementation and knockout by Crispr/Cas9 technology. Sequence analysis showed that a single base mutation (T to A) occurred in the sixth exon of RA1 and resulted in a change from Isoleucine (Ile) to Lysine (Lys). Real-time PCR analyses showed that RA1 expression levels were constitutive in most tissues, but most abundant in the leaves and stems. By transient expression in Nicotiana benthamiana, we found that RA1 protein was localized in the chloroplast. Expression levels of chlorophyll biosynthesis and plastid development related genes were disordered in the ra1 mutant. RNA analysis revealed biogenesis of chloroplast rRNAs was abnormal in ra1. Meanwhile, western blotting showed that synthesis of proteins associated with plastid development was significantly repressed. These results suggest that RA1 is involved in early chloroplast development and establishment of the plastidic ribosome system in rice.

A selfish genetic element confers non-Mendelian inheritance in rice.

Selfish genetic elements are pervasive in eukaryote genomes, but their role remains controversial. We show that qHMS7, a major quantitative genetic locus for hybrid male sterility between wild rice (Oryza meridionalis) and Asian cultivated rice (O. sativa), contains two tightly linked genes [Open Reading Frame 2 (ORF2) and ORF3]. ORF2 encodes a toxic genetic element that aborts pollen in a sporophytic manner, whereas ORF3 encodes an antidote that protects pollen in a gametophytic manner. Pollens lacking ORF3 are selectively eliminated, leading to segregation distortion in the progeny. Analysis of the genetic sequence suggests that ORF3 arose first, followed by gradual functionalization of ORF2 Furthermore, this toxin-antidote system may have promoted the differentiation and/or maintained the genome stability of wild and cultivated rice.

ALR encoding dCMP deaminase is critical for DNA damage repair, cell cycle progression and plant development in rice.

Deoxycytidine monophosphate deaminase (dCMP deaminase, DCD) is crucial to the production of dTTP needed for DNA replication and damage repair. However, the effect of DCD deficiency and its molecular mechanism are poorly understood in plants. Here, we isolated and characterized a rice albinic leaf and growth retardation (alr) mutant that is manifested by albinic leaves, dwarf stature and necrotic lesions. Map-based cloning and complementation revealed that ALR encodes a DCD protein. OsDCD was expressed ubiquitously in all tissues. Enzyme activity assays showed that OsDCD catalyses conversion of dCMP to dUMP, and the ΔDCD protein in the alr mutant is a loss-of-function protein that lacks binding ability. We report that alr plants have typical DCD-mediated imbalanced dNTP pools with decreased dTTP; exogenous dTTP recovers the wild-type phenotype. A comet assay and Trypan Blue staining showed that OsDCD deficiency causes accumulation of DNA damage in the alr mutant, sometimes leading to cell apoptosis. Moreover, OsDCD deficiency triggered cell cycle checkpoints and arrested cell progression at the G1/S-phase. The expression of nuclear and plastid genome replication genes was down-regulated under decreased dTTP, and together with decreased cell proliferation and defective chloroplast development in the alr mutant this demonstrated the molecular and physiological roles of DCD-mediated dNTP pool balance in plant development.

Top Bending Panicle1 is involved in brassinosteroid signaling and regulates the plant architecture in rice.

Brassinosteroids (BRs) regulate several aspects of plant growth and development. Although extensive studies have shown that BR signaling is conservative in higher plants, the molecular mechanism of regulating plant architecture in rice still remains to be explored. Here, we characterized a rice mutant named top bending panicle1 (tbp1). Compared to wild type, tbp1 mutant plants displayed semi-dwarf stature, erect leaves, small and round grains, as well as more tillers. Remarkably, the panicles of tbp1 plants were shorter and denser, and the tops of the panicles were curved by rolling of the base of flag leaves, which was later verified as due to reduced bulliform cell numbers. Map-based cloning, together with transgenic complementation and RNA-interference tests, revealed that TBP1 is a member of the somatic embryogenesis receptor kinases (SERKs) family involved in BR signaling. Furthermore, bimolecular fluorescence complementation and co-immunoprecipitation analysis demonstrated that a substitution at 61st amino acid (His61Leu) in the tbp1 mutant may result in a reduction of the interaction between TBP1 and OsBRI1 (BR receptor in rice). Taken together, our results demonstrate that TBP1 plays a significant role in regulating plant architecture via the brassinosteroid signaling pathway.

The catalytic subunit of magnesium-protoporphyrin IX monomethyl ester cyclase forms a chloroplast complex to regulate chlorophyll biosynthesis in rice.

KEY MESSAGE: YGL8 has the dual functions in Chl biosynthesis: one as a catalytic subunit of MgPME cyclase, the other as a core component of FLU-YGL8-LCAA-POR complex in Chl biosynthesis. Magnesium-protoporphyrin IX monomethyl ester (MgPME) cyclase is an essential enzyme involved in chlorophyll (Chl) biosynthesis. However, its roles in regulating Chl biosynthesis are not fully explored. In this study, we isolated a rice mutant yellow-green leaf 8 (ygl8) that exhibited chlorosis phenotype with abnormal chloroplast development in young leaves. As the development of leaves, the chlorotic plants turned green accompanied by restorations in Chl content and chloroplast ultrastructure. Map-based cloning revealed that the ygl8 gene encodes a catalytic subunit of MgPME cyclase. The ygl8 mutation caused a conserved amino acid substitution (Asn182Ser), which was related to the alterations of Chl precursor content. YGL8 was constitutively expressed in various tissues, with more abundance in young leaves and panicles. Furthermore, we showed that expression levels of some nuclear genes associated with Chl biosynthesis were affected in both the ygl8 mutant and YGL8 RNA interference lines. By transient expression in rice protoplasts, we found that N-terminal 40 amino acid residues were enough to localize the YGL8 protein to chloroplast. In vivo experiments demonstrated a physical interaction between YGL8 and a rice chloroplast protein, low chlorophyll accumulation A (OsLCAA). Moreover, bimolecular fluorescence complementation assays revealed that YGL8 also interacted with the other two rice chloroplast proteins, viz. fluorescent (OsFLU1) and NADPH:protochlorophyllide oxidoreductase (OsPORB). These results provide new insights into the roles of YGL8, not only as a subunit with catalytic activity, but as a core component of FLU-YGL8-LCAA-POR complex required for Chl biosynthesis.