Cholesterylation of Smoothened is a calcium-accelerated autoreaction involving an intramolecular ester intermediate.

Hedgehog (Hh) is a morphogen that binds to its receptor Patched 1 and activates Smoothened (SMO), thereby governing embryonic development and postnatal tissue homeostasis. Cholesterol can bind and covalently conjugate to the luminal cysteine-rich domain (CRD) of human SMO at the D95 residue (D99 in mouse). The reaction mechanism and biological function of SMO cholesterylation have not been elucidated. Here, we show that the SMO-CRD undergoes auto-cholesterylation which is boosted by calcium and involves an intramolecular ester intermediate. In cells, Hh stimulation elevates local calcium concentration in the SMO-localized endosomes through store-operated calcium entry. In addition, we identify the signaling-incompetent SMO D95E mutation, and the D95E mutant SMO can bind cholesterol but cannot be modified or activated by cholesterol. The homozygous SmoD99E/D99E knockin mice are embryonic lethal with severe developmental delay, demonstrating that cholesterylation of CRD is required for full-length SMO activation. Our work reveals the unique autocatalytic mechanism of SMO cholesterylation and an unprecedented role of calcium in Hh signaling.

Integration of miRNAs, Degradome, and Transcriptome Omics Uncovers a Complex Regulatory Network and Provides Insights Into Lipid and Fatty Acid Synthesis During Sesame Seed Development.

Sesame (Sesamum indicum L.) has always been known as a health-promoting oilseed crop because of its nutrient-rich oil. In recent years, studies have focused on lipid and fatty acid (FA) biosynthesis in various plants by high-throughput sequencing. Here, we integrated transcriptomics, small RNAs, and the degradome to establish a comprehensive reserve intensive on key regulatory micro RNA (miRNA)-targeting circuits to better understand the transcriptional and translational regulation of the oil biosynthesis mechanism in sesame seed development. Deep sequencing was performed to differentially express 220 miRNAs, including 65 novel miRNAs, in different developmental periods of seeds. GO and integrated KEGG analysis revealed 32 pairs of miRNA targets with negatively correlated expression profiles, of which 12 miRNA-target pairs were further confirmed by RT-PCR. In addition, a regulatory co-expression network was constructed based on the differentially expressed gene (DEG) profiles. The FAD2, LOC10515945, LOC105161564, and LOC105162196 genes were clustered into groups that regulate the accumulation of unsaturated fatty acid (UFA) biosynthesis. The results provide a unique advanced molecular platform for the study of lipid and FA biosynthesis, and this study may serve as a new theoretical reference to obtain increased levels of UFA from higher-quality sesame seed cultivars and other plants.

Immune-based mutation classification enables neoantigen prioritization and immune feature discovery in cancer immunotherapy.

Genetic mutations lead to the production of mutated proteins from which peptides are presented to T cells as cancer neoantigens. Evidence suggests that T cells that target neoantigens are the main mediators of effective cancer immunotherapies. Although algorithms have been used to predict neoantigens, only a minority are immunogenic. The factors that influence neoantigen immunogenicity are not completely understood. Here, we classified human neoantigen/neopeptide data into three categories based on their TCR-pMHC binding events. We observed a conservative mutant orientation of the anchor residue from immunogenic neoantigens which we termed the "NP" rule. By integrating this rule with an existing prediction algorithm, we found improved performance in neoantigen prioritization. To better understand this rule, we solved several neoantigen/MHC structures. These structures showed that neoantigens that follow this rule not only increase peptide-MHC binding affinity but also create new TCR-binding features. These molecular insights highlight the value of immune-based classification in neoantigen studies and may enable the design of more effective cancer immunotherapies.

[Prime editing creates a novel dimension of plant precise genome editing].

The precise genome editing has not been well established in plants, largely because of the limited frequency of homology recombination and the delivery barrier of donor templates. Recently, Dr. Caixia Gao's group from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, developed a series of plant prime editors (PPEs), which mediats the prime editing in the genomes of rice and wheat. The PPE systems are able to generate all 12 kinds of programmable base substitutions, as well desired multiplex nucleotide substitutions and small deletions or insertions without DNA double-strand breaks, thus providing versatile tools for precise plant genome editing. Herein, we introduce the structure and the editing capacity of the PPEs. The attemp on efficiency enhancements of PPEs and other PPEs are also discussed, which may provide a reference for appropriate application of PPEs in plants and also for continuous optimization of the editing tools.

[Exploration into the simplex reinforcing-reducing manipulation of acupuncture].

Four controversial types of simplex reinforcing-reducing manipulation of acupuncture and their possible meanings were summarized to explore several key elements of reinforcing-reducing manipulation of acupuncture, in addition, the simplex reinforcing-reducing manipulation of acupuncture was classified by single factor. It is concluded that the definition of simplex reinforcing-reducing manipulation of acupuncture should try not to include other non-manipulative elements. According to single factor, it can be divided into: needle-oriented reinforcing-reducing manipulation, twisting reinforcing-reducing manipulation, lifting and interpolating reinforcing-reducing manipulation, fast and slow reinforcing-reducing manipulation, breathing reinforcing-reducing manipulation, opening and closing reinforcing-reducing manipulation. In addition, after considering the effect and principle of number reinforcing-reducing manipulation, it can be considered.

Functional and structural characterization of a ß-glucosidase involved in saponin metabolism from intestinal bacteria.

Saponins are natural glycosides widely used in medicine and the food industry. Although saponin metabolism in human is dependent on intestinal microbes, few involving bacteria enzymes have been identified. We cloned BlBG3, a GH3 ß-glucosidase from Bifidobacterium longum, from human stool. We found that BlBG3 catalyzes the hydrolysis of glycoside furostanol and ginsenoside Rb1 at higher efficiency than other microbial ß-glucosidases. Structural analysis of BlBG3 in complex with d-glucose revealed its three unique loops, which form a deep pocket and participate in substrate binding. To understand how substrate is bound to the pocket, molecular docking was performed and the binding interactions of protobioside with BlBG3 were revealed. Mutational study suggested that R484 and H642 are critical for enzymatic activity. Our study presents the first structural and functional analysis of a saponin-processing enzyme from human microbiota.

The Transcription Factor AtDOF4.7 Is Involved in Ethylene- and IDA-Mediated Organ Abscission in Arabidopsis.

Organ abscission is an important plant developmental process that occurs in response to environmental stress or pathogens. In Arabidopsis, ligand signals, such as ethylene or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), can regulate organ abscission. Previously, we reported that overexpression of AtDOF4.7, a transcription factor gene, directly suppresses the expression of the abscission-related gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 2 (ADPG2), resulting in a deficiency of floral organ abscission. However, the relationship between AtDOF4.7 and abscission pathways still needs to be investigated. In this study, we showed that ethylene regulates the expression of AtDOF4.7, and the peptide ligand, IDA negatively regulates AtDOF4.7 at the transcriptional level. Genetic evidence indicates that AtDOF4.7 and IDA are involved in a common pathway, and a MAPK cascade can phosphorylate AtDOF4.7 in vitro. Further in vivo data suggest that AtDOF4.7 protein levels may be regulated by this phosphorylation. Collectively, our results indicate that ethylene regulates AtDOF4.7 that is involved in the IDA-mediated floral organ abscission pathway.

Identification of a regulatory element responsible for salt induction of rice OsRAV2 through ex situ and in situ promoter analysis.

Salt is a major environmental stress factor that can affect rice growth and yields. Recent studies suggested that members of the AP2/ERF domain-containing RAV (related to ABI3/VP1) TF family are involved in abiotic stress adaptation. However, the transcriptional response of rice RAV genes (OsRAVs) to salt has not yet been fully characterized. In this study, the expression patterns of all five OsRAVs were examined under salt stress. Only one gene, OsRAV2, was stably induced by high-salinity treatment. Further expression profile analyses indicated that OsRAV2 is transcriptionally regulated by salt, but not KCl, osmotic stress, cold or ABA (abscisic acid) treatment. To elucidate the regulatory mechanism of the stress response at the transcriptional level, we isolated and characterized the promoter region of OsRAV2 (P OsRAV2 ). Transgenic analysis indicated that P OsRAV2 is induced by salt stress but not osmotic stress or ABA treatment. Serial 5' deletions and site-specific mutations in P OsRAV2 revealed that a GT-1 element located at position -664 relative to the putative translation start site is essential for the salt induction of P OsRAV2 . The regulatory function of the GT-1 element in the salt induction of OsRAV2 was verified in situ in plants with targeted mutations generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. Taken together, our results indicate that the GT-1 element directly controls the salt response of OsRAV2. This study provides a better understanding of the putative functions of OsRAVs and the molecular regulatory mechanisms of plant genes under salt stress.

Low-Temperature-Induced Expression of Rice Ureidoglycolate Amidohydrolase is Mediated by a C-Repeat/Dehydration-Responsive Element that Specifically Interacts with Rice C-Repeat-Binding Factor 3.

Nitrogen recycling and redistribution are important for the environmental stress response of plants. In non-nitrogen-fixing plants, ureide metabolism is crucial to nitrogen recycling from organic sources. Various studies have suggested that the rate-limiting components of ureide metabolism respond to environmental stresses. However, the underlying regulation mechanism is not well understood. In this report, rice ureidoglycolate amidohydrolase (OsUAH), which is a recently identified enzyme catalyzing the final step of ureide degradation, was identified as low-temperature- (LT) but not abscisic acid- (ABA) regulated. To elucidate the LT regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region of OsUAH (P OsUAH ). Series deletions revealed that a minimal region between -522 and -420 relative to the transcriptional start site was sufficient for the cold induction of P OsUAH . Detailed analyses of this 103-bp fragment indicated that a C-repeat/dehydration-responsive (CRT/DRE) element localized at position -434 was essential for LT-responsive expression. A rice C-repeat-binding factors/DRE-binding proteins 1 (CBFs/DREB1s) subfamily member, OsCBF3, was screened to specifically bind to the CRT/DRE element in the minimal region both in yeast one-hybrid assays and in in vitro gel-shift analysis. Moreover, the promoter could be exclusively trans-activated by the interaction between the CRT/DRE element and OsCBF3 in vivo. These findings may help to elucidate the regulation mechanism of stress-responsive ureide metabolism genes and provide an example of the member-specific manipulation of the CBF/DREB1 subfamily.

Isolation and characterization of three cadmium-inducible promoters from Oryza sativa.

Cadmium (Cd) is an important soil pollutant. Developing genetically engineered crops might be a feasible strategy for Cd decontamination and damage prevention. Both genes and promoters are critical for the effective construction of genetically modified plants. Although many functional genes for Cd tolerance and accumulation have been identified, few reports have focused on plant Cd-inducible promoters. Here, we identified three Cd-inducible genes in the rice genome: two tau class glutathione S-transferase (GSTU) genes, OsGSTU5 and OsGSTU37, and an HSP20/alpha crystallin family protein gene, OsHSP18.6. The promoter sequences were isolated and tested in transgenic rice lines using a GUSplus reporter gene. All of the promoters exhibited low background expression under normal conditions and could be strongly induced by Cd stress. Although their strength was comparable to that of the constitutive OsACTIN promoter under Cd stress, their time-dependent expression patterns under both short- and long-term Cd exposure were markedly different. The responses of the three promoters to other heavy metals were also examined. Furthermore, heavy metal-responsive cis elements in the promoters were computationally analyzed, and regions determining the Cd stress response were analyzed using a series of truncations. Our results indicate that the three Cd-inducible rice promoters described herein could potentially be used in applications aimed at improving heavy metal tolerance in crops or for the bio-monitoring of environmental contamination.

Generation of inheritable and "transgene clean" targeted genome-modified rice in later generations using the CRISPR/Cas9 system.

The CRISPR/Cas9 system is becoming an important genome editing tool for crop breeding. Although it has been demonstrated that target mutations can be transmitted to the next generation, their inheritance pattern has not yet been fully elucidated. Here, we describe the CRISPR/Cas9-mediated genome editing of four different rice genes with the help of online target-design tools. High-frequency mutagenesis and a large percentage of putative biallelic mutations were observed in T0 generations. Nonetheless, our results also indicate that the progeny genotypes of biallelic T0 lines are frequently difficult to predict and that the transmission of mutations largely does not conform to classical genetic laws, which suggests that the mutations in T0 transgenic rice are mainly somatic mutations. Next, we followed the inheritance pattern of T1 plants. Regardless of the presence of the CRISPR/Cas9 transgene, the mutations in T1 lines were stably transmitted to later generations, indicating a standard germline transmission pattern. Off-target effects were also evaluated, and our results indicate that with careful target selection, off-target mutations are rare in CRISPR/Cas9-mediated rice gene editing. Taken together, our results indicate the promising production of inheritable and "transgene clean" targeted genome-modified rice in the T1 generation using the CRISPR/Cas9 system.

Identification and analysis of the mechanism underlying heat-inducible expression of rice aconitase 1.

Respiratory metabolism is an important though poorly understood facet of plant adaptation to stress. Posttranslational modification of aconitase, a component of the tricarboxylic acid cycle (TCA), may be involved in stress tolerance. However, such stress-related transcriptional regulation and its mechanism remain unknown. In this study, we found that expression of the rice Aconitase gene OsACO1 is induced in a time-dependent manner by heat but not other typical abiotic stresses. To analyze the transcriptional regulation mechanism underlying the response to heat, the OsACO1 promoter (POsACO1) was isolated and characterized in transgenic rice. Using qualitative and quantitative analyses, we found that the expression of the GUS reporter gene responded to heat in different tissues and at different stages of development when driven by POsACO1. A series of 5' distal deletions of POsACO1 was generated to delineate the region responsible for heat-induced gene expression. Transient expression analyses in tobacco leaves identified a 322-bp minimal region between -1386 and -1065 as being essential and sufficient for heat-induced expression by POsACO1. We screened for known heat response-related cis-elements in this 322-bp region; however, sequences correlating with heat-induced gene expression were not identified in POsACO1. Therefore, truncations and successive mutagenesis analyses were performed in this 322-bp region. By comparing the activities of promoter fragments and their derivatives, our results indicated that the heat response element resided in a 9-bp region between -1132 and -1124, a sequence that contains a W-box motif. Additional site-directed mutagenesis analyses eliminated the heat response activity of POsACO1 via the W-box element, and an electrophoretic mobility shift assay (EMSA) indicated the binding of POsACO1 by factors in the nuclear extracts of heat-stressed rice seedlings in a W-box-dependent manner. Our results illustrate the expression pattern of a key component of the TCA response to abiotic stress and establish a putative regulatory pathway in the transcriptional modulation of rice respiratory metabolism genes in response to heat.

Isolation and functional characterization of a novel rice constitutive promoter.

KEY MESSAGE: A novel rice constitutive promoter (P OsCon1 ) was isolated. The molecular mechanism of the promoter activity was investigated. P OsCon1 could be used as an alternative constitutive promoter for crop transgenic engineering. Monocot constitutive promoter is an important resource for crop transgenic engineering. In this report, we isolated a novel promoter, Oscon1 promoter (P OsCon1 ), from the 5' upstream region of a constitutively expressed rice gene OsDHAR1. In P OsCon1 ::GUS transgenic rice, we showed that P OsCon1 had a broad expression spectrum in all tested tissues. The expression of the promoter was further analyzed in comparison with the previously characterized strong constitutive promoters. P OsCon1 exhibited comparable activity to OsCc1, OsAct1 or ZmUbi promoters in most tissues, and more active than 35S promoter in roots, seeds, and calli. Further quantitative assays indicated that P OsCon1 activity was not affected by developmental stages or by environmental factors. Further, 5'-deletions analysis indicated that the distinct regions might contribute to the strong expression of P OsCon1 in different tissues. Overall, our results suggest that P OsCon1 is a novel constitutive promoter, which could potentially use in transgenic crop development.

Identification and utilization of cleistogamy gene cl7(t) in rice (Oryza sativa L.).

Gene transformation is an important method for improvement of plants into elite varieties. However, the possibility of gene flow between genetically modified (GM) crops and similar species is a serious public issue that may potentially endanger ecological stability. Cleistogamy is expected to be an ideal genetic tool for preventing transgene propagation from GM crops. A rice mutant, cl7(t), was created by ethyl methanesulfonate mutagenesis. The mutant exhibited cleistogamy, and had closed spikelets, reduced plant height, and altered morphology of the leaves, panicle, and seeds. Anatomical investigations revealed that the cl7(t) mutant contained more vascular bundles and thicker stems than the wild type, which increased the mechanical strength of its internodes, and anti-lodging ability. Further studies demonstrated that the force required to open the lemma and palea was higher in the cl7(t) mutant, and there was weak swelling ability in the lodicules, which leads to cleistogamy. Allelic analyses and complementation tests indicated that cl7(t) was a novel allele of dep2, a mutant that was previously reported to have similar panicle morphology. Sequence analysis showed that cl7(t) had a single nucleotide substitution (C to A) in the third exon that leads to a Ser substitution with a stop codon, giving a truncated DEP2 protein. Quantitative RT-PCR and in situ hybridization tests demonstrated that there was lower CL7(t) expression level in the spikelets and weaker CL7(t) signals in the lodicules of the cl7(t) mutant compared with wild type, which implies that CL7(t) might participate in the development of lodicules. To improve the agronomic traits of cl7(t) to fit the needs of field production, the cl7(t) mutant was crossed with an intermediate-type rice variety named Guanghui102, which bears some important agronomic traits, including increased grain numbers and high rate of seed setting. Through multi-generational pedigree selection, cleistogamy lines with improved economic traits were obtained, which can be used for the selection of ecologically safe GM rice varieties.

Unravelling mitochondrial retrograde regulation in the abiotic stress induction of rice ALTERNATIVE OXIDASE 1 genes.

Mitochondrial retrograde regulation (MRR) is the transduction of mitochondrial signals to mediate nuclear gene expression. It is not clear whether MRR is a common regulation mechanism in plant abiotic stress response. In this study, we analysed the early abiotic stress response of the rice OsAOX1 genes, and the induction of OsAOX1a and OsAOX1b (OsAOX1a/b) was selected as a working model for the stress-induced MRR studies. We found that the induction mediated by the superoxide ion (O2·(-) )-generating chemical methyl viologen was stronger than that of hydrogen peroxide (H2 O2 ). The addition of reactive oxygen species (ROS) scavengers demonstrated that the stress induction was reduced by eliminating O2·(-) . Furthermore, the stress induction did not rely on chloroplast- or cytosol-derived O2·(-) . Next, we generated transgenic plants overexpressing the superoxide dismutase (SOD) gene at different subcellular locations. The results suggest that only the mitochondrial SOD, OsMSD, attenuated the stress induction of OsAOX1a/b specifically. Therefore, our findings demonstrate that abiotic stress initiates the MRR on OsAOX1a/b and that mitochondrial O2·(-) is involved in the process.

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis.

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin-related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin-D-induced depolymerization or phalloidin-induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild-type (WT) Arabidopsis plants. Light-induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.

Regulation of stomatal opening by the guard cell expansin AtEXPA1.

Stomatal movement is strictly regulated by various intracellular and extracellular factors in response environmental signals. In our recent study, we found that an Arabidopsis guard cell expressed expansin, AtEXPA1, regulates stomatal opening by altering the structure of the guard cell wall. This addendum proposes a mechanism by which guard cell expansins regulate stomatal movement.

Overexpression of the Arabidopsis α-expansin gene AtEXPA1 accelerates stomatal opening by decreasing the volumetric elastic modulus.

Guard cell walls of stomata are highly specialized in plants. Previous research focused on the structure and anatomy of guard cell walls, but little is known about guard cell regulation during stomata movement. In this work, we investigate the possible biological role of the Arabidopsis expansin gene AtEXPA1 in stomatal opening. The AtEXPA1 promoter drove the expression of the GUS reporter gene specifically in guard cells. Light-induced stomatal opening was accelerated in 35S::AtEXPA1 lines, whereas the anti-AtEXPA1 antibody decelerated light-induced stomatal opening. The inhibition of the anti-AtEXPA1 antibody on stomatal opening was largely dependent on the environmental pH. The volumetric elastic modulus (ε) was measured as an indicator of changes in the cell wall. The ε value of guard cells in 35S::AtEXPA1 lines was smaller than in the wild types. The putative role of AtEXPA1 as controller of stomatal opening rate and its regulation are discussed.

Overexpression of AtDOF4.7, an Arabidopsis DOF family transcription factor, induces floral organ abscission deficiency in Arabidopsis.

After flower pollination, a programmed process called abscission occurs in which unwanted floral organs are actively shed from the main plant body. We found that a member of the DOF (for DNA binding with one finger) transcription factor family, Arabidopsis (Arabidopsis thaliana) DOF4.7, was expressed robustly in the abscission zone. The Arabidopsis 35S::AtDOF4.7 lines with constitutive expression of AtDOF4.7 exhibited an ethylene-independent floral organ abscission deficiency. In these lines, anatomical analyses showed that the formation of the abscission zone was normal. However, dissolution of the middle lamella failed to separate between the cell walls. AtDOF4.7 was identified as a nucleus-localized transcription factor. This protein had both in vitro and in vivo binding activity to typical DOF cis-elements in the promoter of an abscission-related polygalacturonase (PG) gene, PGAZAT. Overexpression of AtDOF4.7 resulted in down-regulation of PGAZAT. AtDOF4.7 interacted with another abscission-related transcription factor, Arabidopsis ZINC FINGER PROTEIN2. Taken together, our results suggest that AtDOF4.7 participates in the control of abscission as part of the transcription complex that directly regulates the expression of cell wall hydrolysis enzymes.

Array and distribution of actin filaments in guard cells contribute to the determination of stomatal aperture.

Actin filaments in guard cells and their dynamics function in regulating stomatal movement. In this study, the array and distribution of actin filaments in guard cells during stomatal movement were studied with two vital labeling, microinjection of alexa-phalloidin in Vicia faba and expression of GFP-mTn in tobacco. We found that the random array of actin filaments in the most of the closed stomata changed to a ring-like array after stomatal open. And actin filaments, which were throughout the cytoplasm of guard cells of closed stomata (even distribution), were mainly found in the cortical cytoplasm in the case of open stomata (cortical distribution). These results revealed that the random array and even distribution of actin filaments in guard cells may be required for keeping the closed stomata; similarly, the ring-like array and cortical distribution of actin filaments function in sustaining open stomata. Furthermore, we found that actin depolymerization, the trait of moving stomata, facilitates the transformation of actin array and distribution with stomatal movement. So, the depolymerization of actin filaments was favorable for the changes of actin array and distribution in guard cells and thus facilitated stomatal movement.