Haplotype-resolved genome assembly and implementation of VitExpress, an open interactive transcriptomic platform for grapevine.

Haplotype-resolved genome assemblies were produced for Chasselas and Ugni Blanc, two heterozygous Vitis vinifera cultivars by combining high-fidelity long-read sequencing and high-throughput chromosome conformation capture (Hi-C). The telomere-to-telomere full coverage of the chromosomes allowed us to assemble separately the two haplo-genomes of both cultivars and revealed structural variations between the two haplotypes of a given cultivar. The deletions/insertions, inversions, translocations, and duplications provide insight into the evolutionary history and parental relationship among grape varieties. Integration of de novo single long-read sequencing of full-length transcript isoforms (Iso-Seq) yielded a highly improved genome annotation. Given its higher contiguity, and the robustness of the IsoSeq-based annotation, the Chasselas assembly meets the standard to become the annotated reference genome for V. vinifera. Building on these resources, we developed VitExpress, an open interactive transcriptomic platform, that provides a genome browser and integrated web tools for expression profiling, and a set of statistical tools (StatTools) for the identification of highly correlated genes. Implementation of the correlation finder tool for MybA1, a major regulator of the anthocyanin pathway, identified candidate genes associated with anthocyanin metabolism, whose expression patterns were experimentally validated as discriminating between black and white grapes. These resources and innovative tools for mining genome-related data are anticipated to foster advances in several areas of grapevine research.

Bifunctional transcription factors SlERF.H5 and H7 activate cell wall and repress gibberellin biosynthesis genes in tomato via a conserved motif.

The plant cell wall is a dynamic structure that plays an essential role in development, but the mechanism regulating cell wall formation remains poorly understood. We demonstrate that two transcription factors, SlERF.H5 and SlERF.H7, control cell wall formation and tomato fruit firmness in an additive manner. Knockout of SlERF.H5, SlERF.H7, or both genes decreased cell wall thickness, firmness, and cellulose contents in fruits during early development, especially in double-knockout lines. Overexpressing either gene resulted in thicker cell walls and greater fruit firmness with elevated cellulose levels in fruits but severely dwarf plants with lower gibberellin contents. We further identified that SlERF.H5 and SlERF.H7 activate the cellulose biosynthesis gene SlCESA3 but repress the gibberellin biosynthesis gene GA20ox1. Moreover, we identified a conserved LPL motif in these ERFs responsible for their activities as transcriptional activators and repressors, providing insight into how bifunctional transcription factors modulate distinct developmental processes.

A distal enhancer guides the negative selection of toxic glycoalkaloids during tomato domestication.

Steroidal glycoalkaloids (SGAs) are major plant defense metabolites against pests, while they are considered poisonous in food. The genetic basis that guides negative selection of SGAs production during tomato domestication remains poorly understood. Here, we identify a distal enhancer, GAME Enhancer 1 (GE1), as the key regulator of SGAs metabolism in tomato. GE1 recruits MYC2-GAME9 transcriptional complex to regulate the expression of GAME cluster genes via the formation of chromatin loops located in the neighboring DNA region. A naturally occurring GE176 allelic variant is found to be more active in stimulating GAME expression. We show that the weaker GE1 allele has been the main driver for selecting reduced SGAs levels during tomato domestication. Unravelling the "TFs-Enhancer-Promoter" regulatory mechanism operating in SGAs metabolism opens unprecedented prospects for SGAs manipulation in Solanaceae via precision breeding strategies.

A transcriptional cascade mediated by two APETALA2 family members orchestrates carotenoid biosynthesis in tomato.

Carotenoids are important nutrients for human health that must be obtained from plants since they cannot be biosynthesized by the human body. Dissecting the regulatory mechanism of carotenoid metabolism in plants represents the first step toward manipulating carotenoid contents in plants by molecular design breeding. In this study, we determined that SlAP2c, an APETALA2 (AP2) family member, acts as a transcriptional repressor to regulate carotenoid biosynthesis in tomato (Solanum lycopersicum). Knockout of SlAP2c in both the "MicroTom" and "Ailsa Craig" backgrounds resulted in greater lycopene accumulation, whereas overexpression of this gene led to orange-ripe fruit with significantly lower lycopene contents than the wild type. We established that SlAP2c represses the expression of genes involved in lycopene biosynthesis by directly binding to the cis-elements in their promoters. Moreover, SlAP2c relies on its EAR motif to recruit the co-repressors TOPLESS (TPL)2/4 and forms a complex with histone deacetylase (had)1/3, thereby reducing the histone acetylation levels of lycopene biosynthesis genes. Furthermore, SlAP2a, a homolog of SlAP2c, acts upstream of SlAP2c and alleviates the SlAP2c-induced repression of lycopene biosynthesis genes by inhibiting SlAP2c transcription during fruit ripening. Therefore, we identified a transcriptional cascade mediated by AP2 family members that regulates lycopene biosynthesis during fruit ripening in tomato, laying the foundation for the manipulation of carotenoid metabolism in plants.

Ethylene-MPK8-ERF.C1-PR module confers resistance against Botrytis cinerea in tomato fruit without compromising ripening.

The plant hormone ethylene plays a critical role in fruit defense against Botrytis cinerea attack, but the underlying mechanisms remain poorly understood. Here, we showed that ethylene response factor SlERF.C1 acts as a key regulator to trigger the ethylene-mediated defense against B. cinerea in tomato fruits without compromising ripening. Knockout of SlERF.C1 increased fruit susceptibility to B. cinerea with no effect on ripening process, while overexpression enhanced resistance. RNA-Seq, transactivation assays, EMSA and ChIP-qPCR results indicated that SlERF.C1 activated the transcription of PR genes by binding to their promoters. Moreover, SlERF.C1 interacted with the mitogen-activated protein kinase SlMPK8 which allowed SlMPK8 to phosphorylate SlERF.C1 at the Ser174 residue and increases its transcriptional activity. Knocking out of SlMPK8 increased fruit susceptibility to B. cinerea, whereas overexpression enhanced resistance without affecting ripening. Furthermore, genetic crosses between SlMPK8-KO and SlERF.C1-OE lines reduced the resistance to B. cinerea attack in SlERF.C1-OE fruits. In addition, B. cinerea infection induced ethylene production which in turn triggered SlMPK8 transcription and enhanced the phosphorylation of SlERF.C1. Overall, our findings reveal the regulatory mechanism of the 'Ethylene-MPK8-ERF.C1-PR' module in resistance against B. cinerea and provide new insight into the manipulation of gray mold disease in fruits.

Novel lncRNA 803 related to Marek's disease inhibits apoptosis of DF-1 cells.

RESEARCH HIGHLIGHTS: Differentially expressed lncRNAs in spleens of chickens infected with Marek's disease virus at different stages were identified for the first time.The effects of novel lncRNA 803 on p53 pathway and apoptosis of DF-1 cells were reported for the first time.

Gibberellins involved in fruit ripening and softening by mediating multiple hormonal signals in tomato.

The phytohormone ethylene is well known for its important role in the ripening of climacteric fruit, such as tomato (Solanum lycopersicum). However, the role and mode of action of other plant hormones in climacteric fruit ripening regulation are not fully understood. Here, we showed that exogenous GA treatment or increasing endogenous gibberellin content by overexpressing the gibberellin synthesis gene SlGA3ox2 specifically in fruit tissues delayed tomato fruit ripening, whereas treatment with the GA biosynthesis inhibitor paclobutrazol (PAC) accelerated fruit ripening. Moreover, exogenous ethylene treatment cannot completely reverse the delayed fruit ripening phenotype. Furthermore, exogenous GA treatment of ethylene signalling mutant Never ripe (Nr) or SlEBF3-overexpressing lines still delayed fruit ripening, suggesting that GA involved in fruit ripening partially depends on ethylene. Transcriptome profiling showed that gibberellin affect the ripening of fruits by modulating the metabolism and signal transduction of multiple plant hormones, such as auxin and abscisic acid, in addition to ethylene. Overall, the results of this study provide new insight into the regulation of gibberellin in fruit ripening through mediating multiple hormone signals.

SlERF.G3-Like mediates a hierarchical transcriptional cascade to regulate ripening and metabolic changes in tomato fruit.

The tomato ripening process contains complex changes, including ethylene signalling, cell wall softening and numerous metabolic changes. So far, much is still unknown about how tomato plants precisely coordinate fruit maturation and metabolic regulation. In this paper, the ERF family transcription factor SlERF.G3-Like in tomato was found to be involved in the regulation of ethylene synthesis, cell wall degradation and the flavonoid pathway. We show that the master ripening regulator SlRIN was found to directly bind to the promoter region of SlERF.G3-Like to activate its expression. In addition, we managed to increase the production of resveratrol derivatives from ~1.44 mg/g DW in E8:VvStSy line to ~2.43 mg/g DW by crossing p35S: SlERF.G3-Like with the E8:VvStSy line. Our data provide direct evidence that SlERF.G3-Like, a hierarchical transcriptional factor, can directly manipulate pathways in which tomatoes can coordinate fruit maturation and metabolic changes. We also attest that SlERF.G3-Like can be used as an effective tool for phenylpropanoid metabolic engineering.

The novel lncRNA-9802/miR-1646 axis affects cell proliferation of DF-1 by regulating Bax/Bcl-2 signaling pathway.

Marek's disease (MD) is a severe infectious and immunosuppressive neoplastic condition that significantly impacts the global poultry industry. Investigating the role of non-coding RNA in pathogenic mechanisms of MD virus (MDV) offers valuable insights for the effective prevention and management of MD. A higher expression of the novel lncRNA-9802 can be found in spleen tissues of MDV-infected chickens from our prior research, and there is a potential association between lncRNA-9802 and cell proliferation. In this study, we further demonstrated that over-expression of lncRNA-9802 could promote the proliferation of DF-1 cells. It has been established that lncRNA-9802 mediated its effects by binding to miR-1646, and further modulated the expression of the Bax and Bcl-2 genes. Deciphering the role of the recently discovered MD-associated lncRNA-9802/miR-1646 axis provides valuable theoretical basis for decoding the molecular mechanisms underlying MDV pathogenesis.

Understanding the mechanism of red light-induced melatonin biosynthesis facilitates the engineering of melatonin-enriched tomatoes.

Melatonin is a functionally conserved broad-spectrum physiological regulator found in most biological organisms in nature. Enrichment of tomato fruit with melatonin not only enhances its agronomic traits but also provides extra health benefits. In this study, we elucidate the full melatonin biosynthesis pathway in tomato fruit by identifying biosynthesis-related genes that encode caffeic acid O-methyltransferase 2 (SlCOMT2) and N-acetyl-5-hydroxytryptamine-methyltransferases 5/7 (SlASMT5/7). We further reveal that red light supplementation significantly enhances the melatonin content in tomato fruit. This induction relies on the "serotonin-N-acetylserotonin-melatonin" biosynthesis route via the SlphyB2-SlPIF4-SlCOMT2 module. Based on the regulatory mechanism, we design a gene-editing strategy to target the binding motif of SlPIF4 in the promoter of SlCOMT2, which significantly enhances the production of melatonin in tomato fruit. Our study provides a good example of how the understanding of plant metabolic pathways responding to environmental factors can guide the engineering of health-promoting foods.

RIN enhances plant disease resistance via root exudate-mediated assembly of disease-suppressive rhizosphere microbiota.

The RIPENING-INHIBITOR (RIN) transcriptional factor is a key regulator governing fruit ripening. While RIN also affects other physiological processes, its potential roles in triggering interactions with the rhizosphere microbiome and plant health are unknown. Here we show that RIN affects microbiome-mediated disease resistance via root exudation, leading to recruitment of microbiota that suppress the soil-borne, phytopathogenic Ralstonia solanacearum bacterium. Compared with the wild-type (WT) plant, RIN mutants had different root exudate profiles, which were associated with distinct changes in microbiome composition and diversity. Specifically, the relative abundances of antibiosis-associated genes and pathogen-suppressing Actinobacteria (Streptomyces) were clearly lower in the rhizosphere of rin mutants. The composition, diversity, and suppressiveness of rin plant microbiomes could be restored by the application of 3-hydroxyflavone and riboflavin, which were exuded in much lower concentrations by the rin mutant. Interestingly, RIN-mediated effects on root exudates, Actinobacteria, and disease suppression were evident from the seedling stage, indicating that RIN plays a dual role in the early assembly of disease-suppressive microbiota and late fruit development. Collectively, our work suggests that, while plant disease resistance is a complex trait driven by interactions between the plant, rhizosphere microbiome, and the pathogen, it can be indirectly manipulated using "prebiotic" compounds that promote the recruitment of disease-suppressive microbiota.

Extraction technology, components analysis and anti-inflammatory activity in vitro of total flavonoids extract from Artemisia anomala S. Moore.

Artemisia anomala S. Moore exerts many pharmacological activities, including the removing of the blood stasis, relieving of the fever and analgesia, reducing the swelling and dampness. In this study, the extraction technology, chemical compositions and anti-inflammatory effect in vitro and mechanism of total flavonoids extract from Artemisia anomala S. Moore were studied. The optimal yield rate of total flavonoids extract was optimized by single factor experiments and response surface method, and the chemical constituents were analyzed by UPLC-QTOF-MS method; and the anti-inflammatory activity of the extract was evaluated with lipopolysaccharide induced RAW 264.7 cells. The highest extraction rate was 2.02% under these conditions of the concentration of ethanol 50%, the ultrasonic extraction time 30 min, and the ratio of solvent volume to material weight 20:1 (ml/g). In addition, the main components of total flavonoid extract were preliminarily identified and deduced based on mass spectrometry information and relevant literatures, and its stronger anti-inflammatory activity was demonstrated by reducing the phagocytosis, the content of nitric oxide and the level of related cytokines (tumor necrosis factor-α, interleukin-10, interleukin-6). Furthermore, it was further revealed that the anti-inflammatory effect of the extract was closely connected with the activation of TLR4-MyD88-NF-κB signalling pathway. This study indicated that the total flavonoids extract from Artemisia anomala S. Moore may be a better candidate anti-inflammatory natural medicine.

Rational Design of Abasic Site-Containing DNA Triplexes To Bind Small Molecules with Low Nanomolar Dissociation Constants.

De novo design of functional biomacromolecules is of great interest to a wide range of fundamental science and technological applications, including understanding life evolution and biomacromolecular structures, developing novel catalysts, inventing medicines, and exploring high-performance materials. However, it is an extremely challenging task and its success is very limited. It requires a deep understanding of the relationships among the primary sequences, the 3D structures, and the functions of biomacromolecules. Herein, we report a rational, de novo design of a DNA aptamer that can bind melamine with high specificity and high affinity (dissociation constant Kd = 4.4 nM). The aptamer is essentially a DNA triplex, but contains an abasic site, to which the melamine binds. The aptamer-ligand recognition involves hydrogen-bonding, π-π stacking, and electrostatic interactions. This strategy has been further tested by designing aptamers to bind to guanosine. It is conceivable that such a rational strategy, with further development, would provide a general framework for designing functional DNA molecules.

The MADS-box protein SlTAGL1 regulates a ripening-associated SlDQD/SDH2 involved in flavonoid biosynthesis and resistance against Botrytis cinerea in post-harvest tomato fruit.

3-Dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) is a key rate-limiting enzyme that catalyzes the synthesis of the shikimate, which is an important metabolic intermediate in plants and animals. However, the function of SlDQD/SDH family genes in tomato (Solanum lycopersicum) fruit metabolites is still unknown. In the present study, we identified a ripening-associated SlDQD/SDH member, SlDQD/SDH2, that plays a key role in shikimate and flavonoid metabolism. Overexpression of this gene resulted in an increased content of shikimate and flavonoids, while knockout of this gene by CRISPR/Cas9 mediated gene editing led to a significantly lower content of shikimate and flavonoids by downregulation of flavonoid biosynthesis-related genes. Moreover, we showed that SlDQD/SDH2 confers resistance against Botrytis cinerea attack in post-harvest tomato fruit. Dual-luciferase reporter and EMSA assays indicated that SlDQD/SDH2 is a direct target of the key ripening regulator SlTAGL1. In general, this study provided a new insight into the biosynthesis of flavonoid and B. cinerea resistance in fruit tomatoes.

ß-1,3-GLUCANASE10 regulates tomato development and disease resistance by modulating callose deposition.

ß-1,3-Glucanases are considered key regulators responsible for the degradation of callose in plants, yet little is known about the role and mode of action of their encoding genes in tomato (Solanum lycopersicum). In the present study, we identified the ß-1,3-glucanase encoding gene ß-1,3-GLUCANASE10 (SlBG10) and revealed its regulation in tomato pollen and fruit development, seed production, and disease resistance by modulating callose deposition. Compared with wild-type (WT) or SlBG10 overexpressing (SlBG10-OE) lines, knockout of SlBG10 caused pollen arrest and failure to set fruit with reduced male rather than female fecundity. Further analyses showed that SlBG10-knockout promoted callose deposition in anther at the tetrad-to-microspore stages, resulting in pollen abortion and male sterility. Moreover, loss-of-function SlBG10 delayed degradation of endosperm cell wall calloses during cellularization and impeded early seed development. We also uncovered that Botrytis cinerea infection induces SlBG10 expression in WT tomato, and the knockout lines showed increased callose accumulation in fruit pericarps, reduced susceptibility to B. cinerea, and enhanced antioxidant capacity to maintain tomato fruit quality. However, the expression of genes encoding cell wall hydrolases decreased in SlBG10-knockout tomatoes and thus led to an increase in pericarp epidermal thickness, enhancement in fruit firmness, reduction of fruit water loss, and extension of tomato shelf life. These findings not only expand our understanding of the involvement of ß-1,3-glucanases as callose regulators in multiple developmental processes and pathogen resistance but also provide additional insight into the manipulation of multiagronomic traits for targeted tomato breeding.

Enriching adenosine by thymine-rich DNA oligomers.

Adenosine levels are important in various physiological and pathological activities, but detecting them is difficult because of interference from a complex matrix. This study designed a series of DNA oligomers rich in thymine to enrich adenosine. Their binding affinity (Kd range: 1.25-5.0 mM) to adenosine varied based on the DNA secondary structures, with a clamped hairpin structure showing the highest binding affinity. Compared to other designs, this clamped DNA hairpin underwent the least conformational change during adenosine binding. These DNAs also suppressed the precipitation of supersaturated adenine. Taken together, these results suggest that thymine-rich DNAs could be used to enrich and separate adenosine.

A comprehensive metabolic map reveals major quality regulations in red-flesh kiwifruit (Actinidia chinensis).

Kiwifruit (Actinidia chinensis) is one of the popular fruits world-wide, and its quality is mainly determined by key metabolites (sugars, flavonoids, and vitamins). Previous works on kiwifruit are mostly done via a single omics approach or involve only limited metabolites. Consequently, the dynamic metabolomes during kiwifruit development and ripening and the underlying regulatory mechanisms are poorly understood. In this study, using high-resolution metabolomic and transcriptomic analyses, we investigated kiwifruit metabolic landscapes at 11 different developmental and ripening stages and revealed a parallel classification of 515 metabolites and their co-expressed genes into 10 distinct metabolic vs gene modules (MM vs GM). Through integrative bioinformatics coupled with functional genomic assays, we constructed a global map and uncovered essential transcriptomic and transcriptional regulatory networks for all major metabolic changes that occurred throughout the kiwifruit growth cycle. Apart from known MM vs GM for metabolites such as soluble sugars, we identified novel transcription factors that regulate the accumulation of procyanidins, vitamin C, and other important metabolites. Our findings thus shed light on the kiwifruit metabolic regulatory network and provide a valuable resource for the designed improvement of kiwifruit quality.

Genome-wide characterization of the tomato GASA family identifies SlGASA1 as a repressor of fruit ripening.

Gibberellins (GAs) play crucial roles in a wide range of developmental processes and stress responses in plants. However, the roles of GA-responsive genes in tomato (Solanum lycopersicum) fruit development remain largely unknown. Here, we identify 17 GASA (Gibberellic Acid-Stimulated Arabidopsis) family genes in tomato. These genes encode proteins with a cleavable signal peptide at their N terminus and a conserved GASA domain at their C terminus. The expression levels of all tomato GASA family genes were responsive to exogenous GA treatment, but adding ethylene eliminated this effect. Comprehensive expression profiling of SlGASA family genes showed that SlGASA1 follows a ripening-associated expression pattern, with low expression levels during fruit ripening, suggesting it plays a negative role in regulating ripening. Overexpressing SlGASA1 using a ripening-specific promoter delayed the onset of fruit ripening, whereas SlGASA1-knockdown fruits displayed accelerated ripening. Consistent with their delayed ripening, SlGASA1-overexpressing fruits showed significantly reduced ethylene production and carotenoid contents compared to the wild type. Moreover, ripening-related genes were downregulated in SlGASA1-overexpressing fruits but upregulated in SlGASA1-knockdown fruits compared to the wild type. Yeast two-hybrid, co-immunoprecipitation, transactivation, and DNA pull-down assays indicated that SlGASA1 interacts with the key ripening regulator FRUITFULL1 and represses its activation of the ethylene biosynthesis genes ACS2 and ACO1. Our findings shed new light on the role and mode of action of a GA-responsive gene in tomato fruit ripening.

Structural-Based Stability Enhancement of Antisense DNA Oligonucleotides.

Antisense DNA oligonucleotide (AS) technology is a promising approach to regulate gene expression and cellular processes. For example, ASs can be used to capture the overexpressed, oncogenic miRNAs in tumors to suppress tumor growth. Among many challenges faced by AS approach is the degradation of ASs by nucleases under physiological conditions. Elongating the AS lifespan can substantially enhance the functions of AS. The paper reports a simple strategy to increase the stability of ASs. The authors discover that the ASs degrade quickly if their ends are in unpaired, single-stranded form, but much slower if their ends are in paired duplex form. It is conceivable to integrate this strategy with other strategies (such as chemical modification of ASs backbones) to maximally increase the ASs stabilities.

Transition to ripening in tomato requires hormone-controlled genetic reprogramming initiated in gel tissue.

Ripening is the last stage of the developmental program in fleshy fruits. During this phase, fruits become edible and acquire their unique sensory qualities and post-harvest potential. Although our knowledge of the mechanisms that regulate fruit ripening has improved considerably over the past decades, the processes that trigger the transition to ripening remain poorly deciphered. While transcriptomic profiling of tomato (Solanum lycopersicum L.) fruit ripening to date has mainly focused on the changes occurring in pericarp tissues between the Mature Green and Breaker stages, our study addresses the changes between the Early Mature Green and Late Mature Green stages in the gel and pericarp separately. The data showed that the shift from an inability to initiate ripening to the capacity to undergo full ripening requires extensive transcriptomic reprogramming that takes place first in the locular tissues before extending to the pericarp. Genome-wide transcriptomic profiling revealed the wide diversity of transcription factor (TF) families engaged in the global reprogramming of gene expression and identified those specifically regulated at the Mature Green stage in the gel but not in the pericarp, thereby providing potential targets toward deciphering the initial factors and events that trigger the transition to ripening. The study also uncovered an extensive reformed homeostasis for most plant hormones, highlighting the multihormonal control of ripening initiation. Our data unveil the antagonistic roles of ethylene and auxin during the onset of ripening and show that auxin treatment delays fruit ripening via impairing the expression of genes required for System-2 autocatalytic ethylene production that is essential for climacteric ripening. This study unveils the detailed features of the transcriptomic reprogramming associated with the transition to ripening of tomato fruit and shows that the first changes occur in the locular gel before extending to pericarp and that a reformed auxin homeostasis is essential for the ripening to proceed.