An NLR paralog Pit2 generated from tandem duplication of Pit1 fine-tunes Pit1 localization and function.

NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication.

Knockout of miR396 genes increases seed size and yield in soybean.

Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. miR396 genes have been shown to negatively regulate grain size in rice, but whether miR396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean miR396 genes through genome editing with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)12SF01 in the elite soybean cultivar Zhonghuang 302 (ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants (mir396abcd and mir396acfi), and two quintuple mutants (mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfi plants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems. Taken together, our study demonstrated that loss-of-function of miR396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high-yield soybean.

Key transcription factors regulate fruit ripening and metabolite accumulation in tomato.

Fruit ripening is a complex process involving dynamic changes to metabolites and is controlled by multiple factors, including transcription factors (TFs). Several TFs are reportedly essential regulators of tomato (Solanum lycopersicum) fruit ripening. To evaluate the effects of specific TFs on metabolite accumulation during fruit ripening, we combined CRISPR/Cas9-mediated mutagenesis with metabolome and transcriptome analyses to explore regulatory mechanisms. Specifically, we generated various genetically engineered tomato lines that differed regarding metabolite contents and fruit colors. The metabolite and transcript profiles indicated that the selected TFs have distinct functions that control fruit metabolite contents, especially carotenoids and sugars. Moreover, a mutation to ELONGATED HYPOCOTYL5 (HY5) increased tomato fruit fructose and glucose contents by approximately 20% (relative to the wild-type levels). Our in vitro assay showed that HY5 can bind directly to the G-box cis-element in the Sugars Will Eventually be Exported Transporter (SWEET12c) promoter to activate expression, thereby modulating sugar transport. Our findings provide insights into the mechanisms regulating tomato fruit ripening and metabolic networks, providing the theoretical basis for breeding horticultural crops that produce fruit with diverse flavors and colors.

Breeding exceptionally fragrant soybeans for soy milk with strong aroma.

Knockout of the soybean (Glycine max) betaine aldehyde dehydrogenase genes GmBADH1 and GmBADH2 using CRISPR/Cas12i3 enhances the aroma of soybeans. Soy milk made from the gmbadh1/2 double mutant seeds exhibits a much stronger aroma, which consumers prefer; this mutant has potential for enhancing quality in soy-based products.

Dynamic DNA Methylation Regulates Season-Dependent Secondary Metabolism in the New Shoots of Tea Plants.

Plant secondary metabolites are critical quality-conferring compositions of plant-derived beverages, medicines, and industrial materials. The accumulations of secondary metabolites are highly variable among seasons; however, the underlying regulatory mechanism remains unclear, especially in epigenetic regulation. Here, we used tea plants to explore an important epigenetic mark DNA methylation (5mC)-mediated regulation of plant secondary metabolism in different seasons. Multiple omics analyses were performed on spring and summer new shoots. The results showed that flavonoids and theanine metabolism dominated in the metabolic response to seasons in the new shoots. In summer new shoots, the genes encoding DNA methyltransferases and demethylases were up-regulated, and the global CG and CHG methylation reduced and CHH methylation increased. 5mC methylation in promoter and gene body regions influenced the seasonal response of gene expression; the amplitude of 5mC methylation was highly correlated with that of gene transcriptions. These differentially methylated genes included those encoding enzymes and transcription factors which play important roles in flavonoid and theanine metabolic pathways. The regulatory role of 5mC methylation was further verified by applying a DNA methylation inhibitor. These findings highlight that dynamic DNA methylation plays an important role in seasonal-dependent secondary metabolism and provide new insights for improving tea quality.

Metagenomic insights into the diversity of 2,4-dichlorophenol degraders and the cooperation patterns in a bacterial consortium.

2,4-Dichlorophenol, which is largely employed in herbicides and industrial production, is frequently detected in ecosystems and poses risks to human health and environmental safety. Microbial communities are thought to perform better than individual strains in the complete degradation of organic contaminants. However, the synergistic degradation mechanisms of the microbial consortia involved in 2,4-dichlorophenol degradation are still not widely understood. In this study, a bacterial consortium named DCP-2 that is capable of degrading 2,4-dichlorophenol was obtained. Metagenomic analysis, cultivation-dependent functional verification, and co-occurrence network analysis were combined to reveal the primary 2,4-dichlorophenol degraders and the cooperation patterns in the consortium DCP-2. Metagenomic analysis showed that Pseudomonas, Achromobacter, and Pigmentiphaga were the primary degraders for the complete degradation of 2,4-dichlorophenol. Thirty-nine phylogenetically diverse bacterial genera, such as Brucella, Acinetobacter, Aeromonas, Allochromatium and Bosea, were identified as keystone taxa for 2,4-dichlorophenol degradation by keystone taxa analysis of the co-occurrence networks. In addition, a stable synthetic consortium of isolates from DCP-2 was constructed, consisting of Pseudomonas sp. DD-13 and Brucella sp. FZ-1; this synthetic consortium showed superior degradation capability for 2,4-dichlorophenol in both mineral salt medium and wastewater compared with monoculture. The findings provide valuable insights into the practical bioremediation of 2,4-dichlorophenol-contaminated sites.

DNA cytosine methylation dynamics and functional roles in horticultural crops.

Methylation of cytosine is a conserved epigenetic modification that maintains the dynamic balance of methylation in plants under the regulation of methyltransferases and demethylases. In recent years, the study of DNA methylation in regulating the growth and development of plants and animals has become a key area of research. This review describes the regulatory mechanisms of DNA cytosine methylation in plants. It summarizes studies on epigenetic modifications of DNA methylation in fruit ripening, development, senescence, plant height, organ size, and under biotic and abiotic stresses in horticultural crops. The review provides a theoretical basis for understanding the mechanisms of DNA methylation and their relevance to breeding, genetic improvement, research, innovation, and exploitation of new cultivars of horticultural crops.

The role and interaction between transcription factor NAC-NOR and DNA demethylase SlDML2 in the biosynthesis of tomato fruit flavor volatiles.

Flavor-imparting volatile chemicals accumulate as fruits ripen, making major contributions to taste. The NAC transcription factor nonripening (NAC-NOR) and DNA demethylase 2 (SlDML2) are essential for tomato fruit ripening, but details of the potential roles and the relationship between these two regulators in the synthesis of volatiles are lacking. Here, we show substantial reductions in fatty acid and carotenoid-derived volatiles in tomato slnor and sldml2 mutants. An unexpected finding is the redundancy and divergence in volatile profiles, biosynthetic gene expression, and DNA methylation in slnor and sldml2 mutants relative to wild-type tomato fruit. Reduced transcript levels are accompanied by hypermethylation of promoters, including the NAC-NOR target gene lipoxygenase (SlLOXC) that is involved in fatty acid-derived volatile synthesis. Interestingly, NAC-NOR activates SlDML2 expression by directly binding to its promoter both in vitro and in vivo. Meanwhile, reduced NAC-NOR expression in the sldml2 mutant is accompanied by hypermethylation of its promoter. These results reveal a relationship between SlDML2-mediated DNA demethylation and NAC-NOR during tomato fruit ripening. In addition to providing new insights into the metabolic modulation of flavor volatiles, the outcome of our study contributes to understanding the genetics and control of fruit ripening and quality attributes in tomato.

A histone H3K4me1-specific binding protein is required for siRNA accumulation and DNA methylation at a subset of loci targeted by RNA-directed DNA methylation.

In plants, RNA-directed DNA methylation (RdDM) is a well-known de novo DNA methylation pathway that involves two plant-specific RNA polymerases, Pol IV and Pol V. In this study, we discovered and characterized an RdDM factor, RDM15. Through DNA methylome and genome-wide siRNA analyses, we show that RDM15 is required for RdDM-dependent DNA methylation and siRNA accumulation at a subset of RdDM target loci. We show that RDM15 contributes to Pol V-dependent downstream siRNA accumulation and interacts with NRPE3B, a subunit specific to Pol V. We also show that the C-terminal tudor domain of RDM15 specifically recognizes the histone 3 lysine 4 monomethylation (H3K4me1) mark. Structure analysis of RDM15 in complex with the H3K4me1 peptide showed that the RDM15 tudor domain specifically recognizes the monomethyllysine through an aromatic cage and a specific hydrogen bonding network; this chemical feature-based recognition mechanism differs from all previously reported monomethyllysine recognition mechanisms. RDM15 and H3K4me1 have similar genome-wide distribution patterns at RDM15-dependent RdDM target loci, establishing a link between H3K4me1 and RDM15-mediated RdDM in vivo. In summary, we have identified and characterized a histone H3K4me1-specific binding protein as an RdDM component, and structural analysis of RDM15 revealed a chemical feature-based lower methyllysine recognition mechanism.

A novel protein complex that regulates active DNA demethylation in Arabidopsis.

Active DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression. The 5-methylcytosine DNA glycosylase/lyase ROS1 initiates a base-excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at 1 000s of genomic regions in Arabidopsis. How ROS1 is regulated and targeted to specific genomic regions is not well understood. Here, we report the discovery of an Arabidopsis protein complex that contains ROS1, regulates ROS1 gene expression, and likely targets the ROS1 protein to specific genomic regions. ROS1 physically interacts with a WD40 domain protein (RWD40), which in turn interacts with a methyl-DNA binding protein (RMB1) as well as with a zinc finger and homeobox domain protein (RHD1). RMB1 binds to DNA that is methylated in any sequence context, and this binding is necessary for its function in vivo. Loss-of-function mutations in RWD40, RMB1, or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1. Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter, plants mutated in RWD40, RMB1, or RHD1 show increased ROS1 expression. Importantly, ROS1 binding to the ROS1 promoter requires RWD40, RMB1, and RHD1, suggesting that this complex dictates ROS1 targeting to this locus. Our results demonstrate that ROS1 forms a protein complex with RWD40, RMB1, and RHD1, and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.

In vitro fermentation and isolation of heparin-degrading bacteria from human gut microbiota.

Heparin and its derivative are commonly used as injectable anticoagulants in clinical procedures, but possess poor oral bioavailability. To explore the role of gut microbiota in the poor oral effect of heparin, the degradation profiles of heparin on six human gut microbiota were investigated. The heparin-degradation ability varied significantly among individuals. Furthermore, two strains of heparin-degrading bacteria, Bacteroides ovatus A2 and Bacteroides cellulosilyticus B19, were isolated from the gut microbiota of different individuals and the degradation products of the isolates were profiled. The ΔUA2S-GlcNS6S was the major end product with almost no desulfation. 3-O-sulfo group-containing tetrasaccharides were detected, which indicated that the antithrombin binding site was broken and this explained the lost anticoagulant activity of heparin. Collectively, the present study assessed the degradation profiles of heparin by human gut microbiota and provided references for the development of oral administration of heparin from a gut microbiota perspective.

Loss of salt tolerance during tomato domestication conferred by variation in a Na+ /K+ transporter.

Domestication has resulted in reduced salt tolerance in tomato. To identify the genetic components causing this deficiency, we performed a genome-wide association study (GWAS) for root Na+ /K+ ratio in a population consisting of 369 tomato accessions with large natural variations. The most significant variations associated with root Na+ /K+ ratio were identified within the gene SlHAK20 encoding a member of the clade IV HAK/KUP/KT transporters. We further found that SlHAK20 transports Na+ and K+ and regulates Na+ and K+ homeostasis under salt stress conditions. A variation in the coding sequence of SlHAK20 was found to be the causative variant associated with Na+ /K+ ratio and confer salt tolerance in tomato. Knockout mutations in tomato SlHAK20 and the rice homologous genes resulted in hypersensitivity to salt stress. Together, our study uncovered a previously unknown molecular mechanism of salt tolerance responsible for the deficiency in salt tolerance in cultivated tomato varieties. Our findings provide critical information for molecular breeding to improve salt tolerance in tomato and other crops.

Two different fucosylated chondroitin sulfates: Structural elucidation, stimulating hematopoiesis and immune-enhancing effects.

Two fucosylated chondroitin sulfates FCShp and FCSht were isolated from the sea cucumber Holothuria polii and Holothuria tubulosa, respectively. The NMR spectroscopy and HILIC-FTMS methods were applied for their detailed structural characterization. Chemical analysis indicated that the two FCSs all contained a chondroitin sulfate backbone chondroitin sulfate-like core and fucosyl branches of α-L-Fuc2,4S, α-L-Fuc4S or α-L-Fuc3,4S linked to O-3 of glucuronic acid residues. The main branches of FCShp and FCSht were monofucose, and the small amounts of di-, tri- and tetrafucose with α-1,3-linkage type were also detected. Finally, we investigated the immunomodulatory function of FCShp and FCSht in cyclophosphamide (CTX)-induced immunosuppressed mouse models. The results showed that FCShp and FCSht had beneficial effects on hematopoietic function recovery in CTX-induced bone marrow suppression mice. Notably, the α-L-Fuc2,4S was more important to the activity than α-L-Fuc3,4S. These results provided basis for developing the drugs to reduce side effects of chemotherapy.

Fucoidan from sea cucumber Holothuria polii: Structural elucidation and stimulation of hematopoietic activity.

The structural elucidation of polysaccharides is essential for understanding their structure-bioactivity relationship and related drug development. In this study, fucoidan (Fuchp) was extracted and purified from sea cucumber Holothuria polii. Its sulfate content was 39.5 ± 1.4%, and the "weight-average" molecular mass was 103.1 ± 2.8 kDa. The primary structure of Fuchp was clarified using a combination of acid degradation, tandem mass spectrometry, and nuclear magnetic resonance spectroscopy analysis. As a result, Fuchp was found to be composed of a tetrafucose repeating unit [→3-α-l-Fucp-1 â†’ 3-α-l-Fucp2(OSO3-)-1 â†’ 3-α-l-Fucp2(OSO3-)-1 â†’ 3-α-l-Fucp2,4(OSO3-)-1→]. The stimulating hematopoiesis was further evaluated in a mouse model induced by cyclophosphamide. Based on these findings, intraperitoneally administered Fuchp may accelerate the recovery of white blood cells and neutrophils, in which its activity exceeded that of recombinant human granulocyte colony-stimulating factor (rhG-CSF). Meanwhile, in the background of cyclophosphamide-induced immunosuppression, treatment with Fuchp reduces platelet aggregation caused by CTX, so it might have the effect of reducing the risk of thrombosis. Therefore, Fuchp can be exploited as potentially promising stimulator of hematopoiesis in the future.

Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid.

The widely used Streptococcus pyogenes Cas9 (SpCas9) requires NGG as a protospacer adjacent motif (PAM) for genome editing. Although SpCas9 is a powerful genome-editing tool, its use has been limited on the targetable genomic locus lacking NGG PAM. The SpCas9 variants xCas9 and Cas9-NG have been developed to recognize NG, GAA, and GAT PAMs in human cells. Here, we show that xCas9 cannot recognize NG PAMs in tomato, and Cas9-NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes. In addition, we engineered SpCas9 (XNG-Cas9) based on mutations from both xCas9 and Cas9-NG, and found that XNG-Cas9 can efficiently mutagenize endogenous target sites with NG, GAG, GAA, and GAT PAMs in the tomato or Arabidopsis genomes. The PAM compatibility of XNG-Cas9 is the broadest reported to date among Cas9s (SpCas9 and Cas9-NG) active in plant.

Global increase in DNA methylation during orange fruit development and ripening.

DNA methylation is an important epigenetic mark involved in many biological processes. The genome of the climacteric tomato fruit undergoes a global loss of DNA methylation due to active DNA demethylation during the ripening process. It is unclear whether the ripening of other fruits is also associated with global DNA demethylation. We characterized the single-base resolution DNA methylomes of sweet orange fruits. Compared with immature orange fruits, ripe orange fruits gained DNA methylation at over 30,000 genomic regions and lost DNA methylation at about 1,000 genomic regions, suggesting a global increase in DNA methylation during orange fruit ripening. This increase in DNA methylation was correlated with decreased expression of DNA demethylase genes. The application of a DNA methylation inhibitor interfered with ripening, indicating that the DNA hypermethylation is critical for the proper ripening of orange fruits. We found that ripening-associated DNA hypermethylation was associated with the repression of several hundred genes, such as photosynthesis genes, and with the activation of hundreds of genes, including genes involved in abscisic acid responses. Our results suggest important roles of DNA methylation in orange fruit ripening.

The red bayberry genome and genetic basis of sex determination.

Morella rubra, red bayberry, is an economically important fruit tree in south China. Here, we assembled the first high-quality genome for both a female and a male individual of red bayberry. The genome size was 313-Mb, and 90% sequences were assembled into eight pseudo chromosome molecules, with 32 493 predicted genes. By whole-genome comparison between the female and male and association analysis with sequences of bulked and individual DNA samples from female and male, a 59-Kb region determining female was identified and located on distal end of pseudochromosome 8, which contains abundant transposable element and seven putative genes, four of them are related to sex floral development. This 59-Kb female-specific region was likely to be derived from duplication and rearrangement of paralogous genes and retained non-recombinant in the female-specific region. Sex-specific molecular markers developed from candidate genes co-segregated with sex in a genetically diverse female and male germplasm. We propose sex determination follow the ZW model of female heterogamety. The genome sequence of red bayberry provides a valuable resource for plant sex chromosome evolution and also provides important insights for molecular biology, genetics and modern breeding in Myricaceae family.

A group of SUVH methyl-DNA binding proteins regulate expression of the DNA demethylase ROS1 in Arabidopsis.

DNA methylation is typically regarded as a repressive epigenetic marker for gene expression. Genome-wide DNA methylation patterns in plants are dynamically regulated by the opposing activities of DNA methylation and demethylation reactions. In Arabidopsis, a DNA methylation monitoring sequence (MEMS) in the promoter of the DNA demethylase gene ROS1 functions as a methylstat that senses these opposing activities and regulates genome DNA methylation levels by adjusting ROS1 expression. How DNA methylation in the MEMS region promotes ROS1 expression is not known. Here, we show that several Su(var)3-9 homologs (SUVHs) can sense DNA methylation levels at the MEMS region and function redundantly to promote ROS1 expression. The SUVHs bind to the MEMS region, and the extent of binding is correlated with the methylation level of the MEMS. Mutations in the SUVHs lead to decreased ROS1 expression, causing DNA hypermethylation at more than 1,000 genomic regions. Thus, the SUVHs function to mediate the activation of gene transcription by DNA methylation.