Rice Floury Endosperm26 encoding a mitochondrial single-stranded DNA-binding protein is essential for RNA-splicing of mitochondrial genes and endosperm development.

Endosperm, the major storage organ in cereal grains, determines the grain yield and quality. Mitochondria provide the energy for dry matter accumulation, in the endosperm development. Although mitochondrial single-stranded DNA-binding proteins (mtSSBs) play a canonical role in the maintenance of single-stranded mitochondrial DNA, their molecular functions in RNA processing and endosperm development remain obscure. Here, we report a defective rice endosperm mutant, floury endosperm26 (flo26), which develops abnormal starch grains in the endosperm. Map-based cloning and complementation experiments showed that FLO26 allele encodes a mitochondrial single-stranded DNA-binding protein, named as mtSSB1.1. Loss of function of mtSSB1.1 affects the transcriptional level of many mitochondrially-encoded genes and RNA splicing of nad1, a core component of respiratory chain complex I in mitochondria. As a result, dysfunctional mature nad1 led to dramatically decreased complex I activity, thereby reducing ATP production. Our results reveal that mtSSB1.1 plays an important role in the maintenance of mitochondrial function and endosperm development by stabilizing the splicing of mitochondrial RNA in rice.

The RNA binding protein EHD6 recruits the m6A reader YTH07 and sequesters OsCOL4 mRNA into phase-separated ribonucleoprotein condensates to promote rice flowering.

N6-Methyladenosine (m6A) is one of the most abundant modifications of eukaryotic mRNA, but its comprehensive biological functionality remains further exploration. In this study, we identified and characterized a new flowering-promoting gene, EARLY HEADING DATE6 (EHD6), in rice. EHD6 encodes an RNA recognition motif (RRM)-containing RNA binding protein that is localized in the non-membranous cytoplasm ribonucleoprotein (RNP) granules and can bind both m6A-modified RNA and unmodified RNA indiscriminately. We found that EHD6 can physically interact with YTH07, a YTH (YT521-B homology) domain-containing m6A reader. We showed that their interaction enhances the binding of an m6A-modified RNA and triggers relocation of a portion of YTH07 from the cytoplasm into RNP granules through phase-separated condensation. Within these condensates, the mRNA of a rice flowering repressor, CONSTANS-like 4 (OsCOL4), becomes sequestered, leading to a reduction in its protein abundance and thus accelerated flowering through the Early heading date 1 pathway. Taken together, these results not only shed new light on the molecular mechanism of efficient m6A recognition by the collaboration between an RNA binding protein and YTH family m6A reader, but also uncover the potential for m6A-mediated translation regulation through phase-separated ribonucleoprotein condensation in rice.

Collective Neural Dynamics for Sparse Motion Planning of Redundant Manipulators Without Hessian Matrix Inversion.

Redundant manipulators have been widely used in various industries whose applications not only improve production efficiency and reduce manual labor but also promote innovation in robotics and artificial intelligence. Kinematic control plays a fundamental and crucial role in robot control. Over the past few decades, numerous motion control schemes have been proposed and applied to trajectory tracking tasks. However, most of these schemes do not consider the introduction of sparsity into the motion control of redundant manipulators, resulting in excessive joint movements, which not only consume extra energy but also increase the risk of unexpected collisions in complex environments. To solve this problem, we transform the issue of increasing the sparsity into a nonconvex optimization problem. Furthermore, a collective neural dynamics for sparse motion planning (CNDSMP) scheme for motion planning of redundant manipulators is proposed. By incorporating sparsity into the control scheme, the excessive joint movements are minimized, leading to improved efficiency and reduced collision risks. Through simulations, comparisons, and physical experiments, the effectiveness and superiority of the proposed scheme are demonstrated.

Fine-tuning rice heading date through multiplex editing of the regulatory regions of key genes by CRISPR-Cas9.

Heading date (or flowering time) is a key agronomic trait that affects seasonal and regional adaption of rice cultivars. An unoptimized heading date can either not achieve a high yield or has a high risk of encountering abiotic stresses. There is a strong demand on the mild to moderate adjusting the heading date in breeding practice. Genome editing is a promising method which allows more precise and faster changing the heading date of rice. However, direct knock out of major genes involved in regulating heading date will not always achieve a new germplasm with expected heading date. It is still challenging to quantitatively adjust the heading date of elite cultivars with best adaption for broader region. In this study, we used a CRISPR-Cas9 based genome editing strategy called high-efficiency multiplex promoter-targeting (HMP) to generate novel alleles at cis-regulatory regions of three major heading date genes: Hd1, Ghd7 and DTH8. We achieved a series of germplasm with quantitative variations of heading date by editing promoter regions and adjusting the expression levels of these genes. We performed field trials to screen for the best adapted lines for different regions. We successfully expanded an elite cultivar Ningjing8 (NJ8) to a higher latitude region by selecting a line with a mild early heading phenotype that escaped from cold stress and achieved high yield potential. Our study demonstrates that HMP is a powerful tool for quantitatively regulating rice heading date and expanding elite cultivars to broader regions.

Long short-term memory with activation on gradient.

As the number of long short-term memory (LSTM) layers increases, vanishing/exploding gradient problems exacerbate and have a negative impact on the performance of the LSTM. In addition, the ill-conditioned problem occurs in the training process of LSTM and adversely affects its convergence. In this work, a simple and effective method of the gradient activation is applied to the LSTM, while empirical criteria for choosing gradient activation hyperparameters are found. Activating the gradient refers to modifying the gradient with a specific function named the gradient activation function. Moreover, different activation functions and different gradient operations are compared to prove that the gradient activation is effective on LSTM. Furthermore, comparative experiments are conducted, and their results show that the gradient activation alleviates the above problems and accelerates the convergence of the LSTM. The source code is publicly available at https://github.com/LongJin-lab/ACT-In-NLP.

Activated Gradients for Deep Neural Networks.

Deep neural networks often suffer from poor performance or even training failure due to the ill-conditioned problem, the vanishing/exploding gradient problem, and the saddle point problem. In this article, a novel method by acting the gradient activation function (GAF) on the gradient is proposed to handle these challenges. Intuitively, the GAF enlarges the tiny gradients and restricts the large gradient. Theoretically, this article gives conditions that the GAF needs to meet and, on this basis, proves that the GAF alleviates the problems mentioned above. In addition, this article proves that the convergence rate of SGD with the GAF is faster than that without the GAF under some assumptions. Furthermore, experiments on CIFAR, ImageNet, and PASCAL visual object classes confirm the GAF's effectiveness. The experimental results also demonstrate that the proposed method is able to be adopted in various deep neural networks to improve their performance. The source code is publicly available at https://github.com/LongJin-lab/Activated-Gradients-for-Deep-Neural-Networks.

Tetrapyrrole biosynthesis pathway regulates plastid-to-nucleus signaling by controlling plastid gene expression in plants.

Plastid-to-nucleus retrograde signaling coordinates nuclear gene expression with chloroplast developmental status and is essential for the photoautotrophic lifestyle of plants. Previous studies have established that tetrapyrrole biosynthesis (TPB) and plastid gene expression (PGE) play essential roles in plastid retrograde signaling during early chloroplast biogenesis; however, their functional relationship remains unknown. In this study, we generated a series of rice TPB-related gun (genome uncoupled) mutants and systematically analyzed their effects on nuclear and plastid gene expression under normal conditions or when subjected to treatments with norflurazon (NF; a noncompetitive inhibitor of carotenoid biosynthesis) and/or lincomycin (Lin; a specific inhibitor of plastid translation). We show that under NF treatment, expression of plastid-encoded polymerase (PEP)-transcribed genes is significantly reduced in the wild type but is derepressed in the TPB-related gun mutants. We further demonstrate that the derepressed expression of PEP-transcribed genes may be caused by increased expression of the PEP core subunit and nuclear-encoded sigma factors and by elevated copy numbers of plastid genome per haploid genome. In addition, we show that expression of photosynthesis-associated nuclear genes (PhANGs) and PEP-transcribed genes is correlated in the rice TPB-related gun mutants, with or without NF or Lin treatment. A similar correlation between PhANGs and PGE is also observed in the Arabidopsis gun4 and gun5 mutants. Moreover, we show that increased expression of PEP-transcribed plastid genes is necessary for the gun phenotype in NF-treated TPB-related gun mutants. Further, we provide evidence that these TPB-related GUN genes act upstream of GUN1 in the regulation of retrograde signaling. Taken together, our results suggest that the TPB-related GUN genes control retrograde plastid signaling by regulating the PGE-dependent retrograde signaling pathway.

The N6-methyladenosine binding proteins YTH03/05/10 coordinately regulate rice plant height.

N6-methyladenosine (m6A) is the most widely distributed and most abundant type of mRNA modification in eukaryotic. It provides a posttranscriptional level regulation of gene expression by regulating pre-mRNA splicing, mRNA degradation, or mRNA translational efficiency etc. The function of m6A modification is decoded by binding proteins that can specially bind to m6A. YT521-B homology (YTH) family proteins are the most important m6A-binding proteins in mammals and Arabidopsis. However, their roles in growth and development remain unknown. Here, we demonstrated that the YTH family proteins YTH03, YTH05 and YTH10 specifically bind to m6A-containing RNAs. Knockout of YTH03, YTH05 or YTH10 causes reduced plant height. Further research showed that simultaneously knockout of YTH03, YTH05 and YTH10 shows severe dwarf phenotype, suggesting these three genes regulate rice plant height in a functionally redundant manner. Additional transcriptome study showed that the reduced plant height of the yth03/05/10 triple mutant may be due to the blocked of diterpenoid and brassinolide synthesis pathway. Overall, we demonstrate that YTH03, YTH05 and YTH10 are all the m6A readers in rice and redundantly regulate rice plant height through the hormonal related pathway.

miR-2188-5p promotes GCRV replication by the targeted degradation of klf2a in Ctenopharyngodon idellus.

Studies on host immunity evasion by aquatic viruses have largely focused on coding genes. There is accumulating evidence for the important biological functions of non-coding miRNAs in virus-host interactions. The regulatory functions of non-coding miRNAs in fish reovirus-host interactions remain unknown. Here, miR-2188-5p in grass carp (Ctenopharyngodon idellus), a miRNA specific to teleosts, was predicted to target the 3' UTR of the transcription factor klf2a. A correlation analysis and dual-luciferase reporter assay revealed that miR-2188-5p could induce the degradation of klf2a. The expression of miR-2188-5p induced the degradation of klf2a in a dose-dependent manner, suppressing the type I interferon response and promoting grass carp reovirus (GCRV) replication. As determined by a co-expression analysis, klf2a inhibited viral infection when miR-2188-5p was overexpressed. The targeted degradation of klf2a by miR-2188-5p could inhibit the type I interferon response and promote the replication of GCRV; however, this targeted degradation ability was insufficient to fully inhibit GCRV infection. These results provide novel insights into the regulatory effects and biological functions of non-coding miRNAs in fish-virus interactions.

Krüppel-like factor 2a (KLF2A) suppresses GCRV replication by upregulating serpinc1 expression in Ctenopharyngodon idellus kidney (CIK) cells.

Krüppel-like factor 2a (KLF2A), a transcription factor of the krüppel-like family, is involved in regulating the immune molecules and is associated with viral infection. However, the function of KLF2A during viral infections in fish remains unclear. In this study, grass carp (Ctenopharyngodon idellus) was used to predict the target genes regulated by KLF2A. The results showed that the candidate target genes included four members of the serpin gene family (serpinb1l2, serpinc1, serpinh1a, and serpinh1b). Dual-luciferase experiments showed that klf2a positively regulates serpinc1 expression. Dose-dependent klf2a overexpression in C. idellus kidney (CIK) cells significantly upregulated the expression of serpinc1. Overexpressing klf2a or serpinc1 in CIK cells activated interferon responses and suppressed grass carp reovirus (GCRV) replication. Klf2a and serpinc1 co-expression inhibited GCRV replication. These results show that klf2a upregulates serpinc1 mRNA expression, promotes type 1 interferon responses, and suppresses GCRV infection. This study provides insights into the regulatory role and biological functions of KLF2A in host-virus interactions in fish.

Grass carp SERPINA1 inhibits GCRV infection through degrading CF2.

SERPINA1, a member of the serine protease inhibitor family, plays a role in viral infection and inflammation by regulating the activities of serine and cysteine proteases. To date, there have been no reports on the immune function of SERPINA1 in fishes. In this study, we first cloned the serpina1 gene of grass carp (Ctenopharyngodon idellus) and found that it could respond rapidly to the infection of Grass carp reovirus (GCRV), and overexpression of serpina1 could enhance the antiviral response of CIK cells. A polyclonal antibody of SERPINA1 was prepared, and the protein interacting with SERPINA1 was screened by CoIP/MS in grass carp hepatopancreas tissue. It was found that SERPINA1 interacted with coagulation factor 2 (CF2) and could degrade it in a dose-dependent manner. In addition, overexpression of cf2 contributed to the infection of GCRV in CIK cells, whereas co-expression of serpina1 and cf2 in grass carp reduced the copy number of GCRV in cells. The results showed that grass carp SERPINA1 could inhibit GCRV infection by degrading CF2. This study proposes that SERPINA1 can inhibit viral infection through interaction with the coagulation factor, providing new insights into the molecular mechanism of SERPINA1's antiviral function.

Screening of Genes Related to Sex Determination and Differentiation in Mandarin Fish (Siniperca chuatsi).

Mandarin fish has an XX/XY sex-determination system. The female mandarin fish is typically larger than the male. Sex identification and the discovery of genes related to sex determination in mandarin fish have important theoretical significance in the elucidation of the regulation and evolutionary mechanism of animal reproductive development. In this study, the chromosome-level genome of a female mandarin fish was assembled, and we found that LG24 of the genome was an X chromosome. A total of 61 genes on the X chromosome showed sex-biased expression. Only six gonadal genes (LG24G00426, LG24G003280, LG24G003300, LG24G003730, LG24G004200, and LG24G004770) were expressed in the testes, and the expression of the other gene LG24G003870 isoform 1 in the ovaries was significantly higher than that in the testes (p < 0.01). Five (except LG24G003280 and LG24G003300) of the seven aforementioned genes were expressed at the embryonic development stage, suggesting their involvement in early sex determination. The expression of LG24G004770 (encoding HS6ST 3-B-like) was also significantly higher in female muscles than in male muscles (p < 0.01), indicating other functions related to female growth. ZP3 encoded by LG24G003870 isoform 1 increased the C-terminal transmembrane domain, compared with that encoded by other fish zp3 isoforms, indicating their different functions in sex determination or differentiation. This study provides a foundation for the identification of sex-determining genes in mandarin fish.

Formation-containment control of multi-agent systems with communication delays.

This paper designs formation-containment control algorithms for a class of second-order nonlinear multi-agent systems governed by Euler-Lagrange dynamics with communication delays. The formation-containment problem consists of leader agents' formation control and follower agents' containment control. Firstly, to make the leaders form a desired formation and move collectively with a constant velocity, a coordinated formation control algorithm is designed and the variable-gain technique is used to eliminate the effect of communication delays on the leaders' formation control. Secondly, considering that only the leaders have access to the desired moving velocity, we propose distributed velocity estimators for followers in which the communication delays also exist in the followers' information interaction. By using the estimated velocity information, coordinated containment control laws are designed for the followers to drive them asymptotically converge to the convex hull spanned by all leaders. Furthermore, to increase the system robustness against uncertainties and external disturbances, the adaptive updating laws are designed for all agents. Finally, simulations are given to demonstrate these obtained results.

Characterization of SR-B2a and SR-B2b genes and their ability to promote GCRV infection in grass carp (Ctenopharyngodon idellus).

Scavenger receptor class B type 2 (SR-B2) is a pattern recognition receptor involved in innate immunity in mammals; however, the immunological function of SR-Bs in fish remains unclear. In this study, the full-length cDNA sequences of SR-B2a and SR-B2b from grass carp (Ctenopharyngodon idellus) were cloned and designated as CiSR-B2a and CiSR-B2b. Multiple alignments and phylogenetic analyses deduced that CiSR-B2a and CiSR-B2b had the highest evolutionary conservation and were closely related to the zebrafish (Danio rerio) homologs, DrSR-B2a and DrSR-B2b, respectively. Both CiSR-B2a and CiSR-B2b were expressed in all the tested tissues, with the highest expression levels found in the hepatopancreas. In Ctenopharyngodon idellus kidney cells (CIK), CiSR-B2a and CiSR-B2b were mainly located in the cytoplasm, and a small amount located on the plasma membrane. After challenge with Grass Carp Reovirus (GCRV), the expression of CiSR-B2a and CiSR-B2b were significantly upregulated in the spleen (about 10.27 and 27.19 times higher than that at 0 day, p < 0.01). With CiSR-B2a or CiSR-B2b overexpressed in CIK, the relative copy number of GCRV in the cells was both significantly increased compared to that in the control group, indicating that CiSR-B2a and CiSR-B2b may be important proteins during the infection processes of GCRV.

A functional chromogen gene C from wild rice is involved in a different anthocyanin biosynthesis pathway in indica and japonica.

KEY MESSAGE: we identified a functional chromogen gene C from wild rice, providing a new insight of anthocyanin biosynthesis pathway in indica and japonica. Accumulation of anthocyanin is a desirable trait to be selected in rice domestication, but the molecular mechanism of anthocyanin biosynthesis in rice remains largely unknown. In this study, a novel allele of chromogen gene C, OrC1, from Oryza rufipongon was cloned and identified as a determinant regulator of anthocyanin biosynthesis. Although OrC1 functions in purple apiculus, leaf sheath and stigma in indica background, it only promotes purple apiculus in japonica. Transcriptome analysis revealed that OrC1 regulates flavonoid biosynthesis pathway and activates a few bHLH and WD40 genes of ternary MYB-bHLH-WD40 complex in indica. Differentially expressed genes and metabolites were found in the indica and japonica backgrounds, indicating that OrC1 activated the anthocyanin biosynthetic genes OsCHI, OsF3H and OsANS and produced six metabolites independently. Artificial selection and domestication of C1 gene in rice occurred on the coding region in the two subspecies independently. Our results reveal the regulatory system and domestication of C1, provide new insights into MYB transcript factor involved in anthocyanin biosynthesis, and show the potential of engineering anthocyanin biosynthesis in rice.

Genome-wide identification, evolution of Krüppel-like factors (klfs) and their expressions during GCRV challenge in grass carp (Ctenopharyngodonidella).

The Krüppel-like factors (KLFs) are a family of transcription factors containing three highly conserved tandem zinc finger structures, and each member participates in multiple physiological and pathological processes. The publication of genome sequences and the application of bioinformatics tools have led to the discovery of numerous gene families in fishes. Here, 24 klf genes were re-annotated in grass carp. Subsequently, the number of klf family members were investigated in some representative vertebrate species. Then, a series of bioinformatics analysis showed that grass carp klfs in the same subfamily had similar genome structure patterns and conserved distribution patterns of motifs, which supported their molecular evolutionary relationships. Furthermore, the mRNA expression profiles showed that 24 grass carp klfs were ubiquitously expressed in 11 different tissues, and some of them displayed tissue-enriched expression patterns. Finally, the expressions of the evolutionarily expanded klf members (klf2a, 2b, 2l, 5a, 5b, 5l, 6a, 6b, 7a, 7b, 11a, 11b, 12a, 12b, 15 and 15l) during GCRV infection were also analyzed. The results suggested that grass carp klf genes with common evolutionary sources may share functional diversity and conservation. In conclusion, this study provides preliminary clues for further researches on grass carp klf members and their underlying transcriptional regulatory mechanisms during GCRV infection.

white panicle2 encoding thioredoxin z, regulates plastid RNA editing by interacting with multiple organellar RNA editing factors in rice.

Thioredoxins (TRXs) occur in plant chloroplasts as complex disulphide oxidoreductases. Although many biological processes are regulated by thioredoxins, the regulatory mechanism of chloroplast TRXs are largely unknown. Here we report a rice white panicle2 mutant caused by a mutation in the thioredoxin z gene, an orthologue of AtTRX z in Arabidopsis. white panicle2 (wp2) seedlings exhibited a high-temperature-sensitive albinic phenotype. We found that plastid multiple organellar RNA editing factors (MORFs) were the regulatory targets of thioredoxin z. We showed that OsTRX z protein physically interacts with OsMORFs in a redox-dependent manner and that the redox state of a conserved cysteine in the MORF box is essential for MORF-MORF interactions. wp2 and OsTRX z knockout lines show reduced editing efficiencies in many plastidial-encoded genes especially under high-temperature conditions. An Arabidopsis trx z mutant also exhibited significantly reduced chloroplast RNA editing. Our combined results suggest that thioredoxin z regulates chloroplast RNA editing in plants by controlling the redox state of MORFs.

OsMFS1/OsHOP2 Complex Participates in Rice Male and Female Development.

Meiosis plays an essential role in the production of gametes and genetic diversity of posterities. The normal double-strand break (DSB) repair is vital to homologous recombination (HR) and occurrence of DNA fragment exchange, but the underlying molecular mechanism remain elusive. Here, we characterized a completely sterile Osmfs1 (male and female sterility 1) mutant which has its pollen and embryo sacs both aborted at the reproductive stage due to severe chromosome defection. Map-based cloning revealed that the OsMFS1 encodes a meiotic coiled-coil protein, and it is responsible for DSB repairing that acts as an important cofactor to stimulate the single strand invasion. Expression pattern analyses showed the OsMFS1 was preferentially expressed in meiosis stage. Subcellular localization analysis of OsMFS1 revealed its association with the nucleus exclusively. In addition, a yeast two-hybrid (Y2H) and pull-down assay showed that OsMFS1 could physically interact with OsHOP2 protein to form a stable complex to ensure faithful homologous recombination. Taken together, our results indicated that OsMFS1 is indispensable to the normal development of anther and embryo sacs in rice.

Rice OsBT1 regulates seed dormancy through the glycometabolism pathway.

Seed dormancy and germination in rice (Oryza sativa L.) are complex and important agronomic traits that involve a number of physiological processes and energy. A mutant named h470 selected from a60Co-radiated indica cultivar N22 population had weakened dormancy that was insensitive to Gibberellin (GA) and Abscisic acid (ABA). The levels of GA4 and ABA were higher in h470 than in wild-type (WT) plants. The gene controlling seed dormancy in h470 was cloned by mut-map and transgenesis and confirmed to encode an ADP-glucose transporter protein. A 1 bp deletion in Os02g0202400 (OsBT1) caused the weaker seed dormancy in h470. Metabolomics analyses showed that most sugar components were higher in h470 seeds than the wild type. The mutation in h470 affected glycometabolism.