Genomes of autotetraploid wild and cultivated Ziziphus mauritiana reveal polyploid evolution and crop domestication.

Indian jujube (Ziziphus mauritiana) holds a prominent position in the global fruit and pharmaceutical markets. Here, we report the assemblies of haplotype-resolved, telomere-to-telomere genomes of autotetraploid wild and cultivated Indian jujube plants using a two-stage assembly strategy. The generation of these genomes permitted in-depth investigations into the divergence and evolutionary history of this important fruit crop. Using a graph-based pan-genome constructed from eight monoploid genomes, we identified structural variation (SV)-FST hotspots and SV hotspots. Gap-free genomes provide a means to obtain a global view of centromere structures. We identified presence-absence variation-related genes in four monoploid genomes (cI, cIII, wI, and wIII) and resequencing populations. We also present the population structure and domestication trajectory of the Indian jujube based on the resequencing of 73 wild and cultivated accessions. Metabolomic and transcriptomic analyses of mature fruits of wild and cultivated accessions unveiled the genetic basis underlying loss of fruit astringency during domestication of Indian jujube. This study reveals mechanisms underlying the divergence, evolution, and domestication of the autotetraploid Indian jujube and provides rich and reliable genetic resources for future research.

H2A mono-ubiquitination differentiates FACT's functions in nucleosome assembly and disassembly.

The histone chaperone FACT (FAcilitates Chromatin Transcription) plays an essential role in transcription and DNA replication by its dual functions on nucleosome assembly to maintain chromatin integrity and nucleosome disassembly to destabilize nucleosome and facilitate its accessibility simultaneously. Mono-ubiquitination at Lysine 119 of H2A (ubH2A) has been suggested to repress transcription by preventing the recruitment of FACT at early elongation process. However, up to date, how ubH2A directly affects FACT on nucleosome assembly and disassembly remains elusive. In this study, we demonstrated that the dual functions of FACT are differently regulated by ubH2A. The H2A ubiquitination does not affect FACT's chaperone function in nucleosome assembly and FACT can deposit ubH2A-H2B dimer on tetrasome to form intact nucleosome. However, ubH2A greatly restricts FACT binding on nucleosome and inhibits its activity of nucleosome disassembly. Interestingly, deubiquitination of ubH2A rescues the nucleosome disassembly function of FACT to activate gene transcription. Our findings provide mechanistic insights of how H2A ubiquitination affects FACT in breaking nucleosome and maintaining its integrity, which sheds light on the biological function of ubH2A and various FACT's activity under different chromatin states.

Jasmonate-based warfare between the pathogenic intruder and host plant: who wins?

Plants and microbial pathogens often engage in a fierce war that determines their survival. Host plants have evolved sophisticated regulatory mechanisms to fine-tune defense responses to counter attacks from pathogens, while pathogens often hijack the lipid-derived phytohormone jasmonate to cause hormonal signaling imbalances for efficient infection. This review focuses on the jasmonate-based warfare between host plants and pathogenic intruders, and further discusses approaches to uncouple plant growth and defense tradeoffs in crop breeding.

Strigolactone mimic 2-nitrodebranone is highly active in Arabidopsis growth and development.

Strigolactones play crucial roles in regulating plant architecture and development, as endogenous hormones, and orchestrating symbiotic interactions with fungi and parasitic plants, as components of root exudates. rac-GR24 is currently the most widely used strigolactone analog and serves as a reference compound in investigating the action of strigolactones. In this study, we evaluated a suite of debranones and found that 2-nitrodebranone (2NOD) exhibited higher biological activity than rac-GR24 in various aspects of plant growth and development in Arabidopsis, including hypocotyl elongation inhibition, root hair promotion and senescence acceleration. The enhanced activity of 2NOD in promoting AtD14-SMXL7 and AtD14-MAX2 interactions indicates that the molecular structure of 2NOD is a better match for the ligand perception site pocket of D14. Moreover, 2NOD showed lower activity than rac-GR24 in promoting Orobanche cumana seed germination, suggesting its higher ability to control plant architecture than parasitic interactions. In combination with the improved stability of 2NOD, these results demonstrate that 2NOD is a strigolactone analog that can specifically mimic the activity of strigolactones and that 2NOD exhibits strong potential as a tool for studying the strigolactone signaling pathway in plants.

DWARF14 is a non-canonical hormone receptor for strigolactone.

Classical hormone receptors reversibly and non-covalently bind active hormone molecules, which are generated by biosynthetic enzymes, to trigger signal transduction. The α/ß hydrolase DWARF14 (D14), which hydrolyses the plant branching hormone strigolactone and interacts with the F-box protein D3/MAX2, is probably involved in strigolactone detection. However, the active form of strigolactone has yet to be identified and it is unclear which protein directly binds the active form of strigolactone, and in which manner, to act as the genuine strigolactone receptor. Here we report the crystal structure of the strigolactone-induced AtD14-D3-ASK1 complex, reveal that Arabidopsis thaliana (At)D14 undergoes an open-to-closed state transition to trigger strigolactone signalling, and demonstrate that strigolactone is hydrolysed into a covalently linked intermediate molecule (CLIM) to initiate a conformational change of AtD14 to facilitate interaction with D3. Notably, analyses of a highly branched Arabidopsis mutant d14-5 show that the AtD14(G158E) mutant maintains enzyme activity to hydrolyse strigolactone, but fails to efficiently interact with D3/MAX2 and loses the ability to act as a receptor that triggers strigolactone signalling in planta. These findings uncover a mechanism underlying the allosteric activation of AtD14 by strigolactone hydrolysis into CLIM, and define AtD14 as a non-canonical hormone receptor with dual functions to generate and sense the active form of strigolactone.

The genomic architecture of the sex-determining region and sex-related metabolic variation in Ginkgobiloba.

Sex differences and evolutionary differences are critical biological issues. Ginkgo is an ancient lineage of dioecious gymnosperms with special value for studying the mechanism of sex determination in plants. However, the major genetic basic underlying sex chromosomes remains to be uncovered. In this study, we identify the sex-determining region of Ginkgo and locate it to the area from megabases 48 to 75 on chromosome 2. We find that the male sex-determining region of Ginkgo contains more than 200 genes, including four MADS-box genes, demonstrating that the Ginkgo sex determination system is of the XY type. We also find that genetic sex differences result in specialized flavonoid metabolism and regulation in each sex. These findings establish a foundation for revealing the molecular mechanism of sexual dimorphism and promoting the development of the Ginkgo industry.

Efficient ASK-assisted system for expression and purification of plant F-box proteins.

Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins.

Rice DWARF14 acts as an unconventional hormone receptor for strigolactone.

Strigolactones (SLs) act as an important class of phytohormones to regulate plant shoot branching, and also serve as rhizosphere signals to mediate interactions of host plants with soil microbes and parasitic weeds. SL receptors in dicots, such as DWARF14 in Arabidopsis (AtD14), RMS3 in pea, and ShHTL7 in Striga, serve as unconventional receptors that hydrolyze SLs into a D-ring-derived intermediate CLIM and irreversibly bind CLIM to trigger SL signal transduction. Here, we show that D14 from the monocot rice can complement Arabidopsis d14 mutant and interact with the SL signaling components in Arabidopsis. Our results further reveal that rice D14, similar to SL receptors in dicots, also serves as an unconventional hormone receptor that generates and irreversibly binds the active form of SLs. These findings uncover the conserved functions of D14 proteins in monocots and dicots.

Endogenous Bioactive Jasmonate Is Composed of a Set of (+)-7-iso-JA-Amino Acid Conjugates.

Jasmonates (JAs) regulate a wide range of plant defense and development processes. The bioactive JA is perceived by its receptor COI1 to trigger the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins and subsequently derepress the JAZ-repressed transcription factors for activation of expression of JA-responsive genes. So far, (+)-7-iso-JA-l-Ile has been the only identified endogenous bioactive JA molecule. Here, we designed coronafacic acid (CFA) conjugates with all the amino acids (CFA-AA) to mimic the JA amino acid conjugates, and revealed that (+)-7-iso-JA-Leu, (+)-7-iso-JA-Val, (+)-7-iso-JA-Met, and (+)-7-iso-JA-Ala are new endogenous bioactive JA molecules. Furthermore, our studies uncover the general characteristics for all the bioactive JA molecules, and provide a new strategy to synthetically generate novel active JA molecules.

Arabidopsis DELLA and JAZ proteins bind the WD-repeat/bHLH/MYB complex to modulate gibberellin and jasmonate signaling synergy.

Integration of diverse environmental and endogenous signals to coordinately regulate growth, development, and defense is essential for plants to survive in their natural habitat. The hormonal signals gibberellin (GA) and jasmonate (JA) antagonistically and synergistically regulate diverse aspects of plant growth, development, and defense. GA and JA synergistically induce initiation of trichomes, which assist seed dispersal and act as barriers to protect plants against insect attack, pathogen infection, excessive water loss, and UV irradiation. However, the molecular mechanism underlying such synergism between GA and JA signaling remains unclear. In this study, we revealed a mechanism for GA and JA signaling synergy and identified a signaling complex of the GA pathway in regulation of trichome initiation. Molecular, biochemical, and genetic evidence showed that the WD-repeat/bHLH/MYB complex acts as a direct target of DELLAs in the GA pathway and that both DELLAs and JAZs interacted with the WD-repeat/bHLH/MYB complex to mediate synergism between GA and JA signaling in regulating trichome development. GA and JA induce degradation of DELLAs and JASMONATE ZIM-domain proteins to coordinately activate the WD-repeat/bHLH/MYB complex and synergistically and mutually dependently induce trichome initiation. This study provides deep insights into the molecular mechanisms for integration of different hormonal signals to synergistically regulate plant development.

The Arabidopsis F-box protein CORONATINE INSENSITIVE1 is stabilized by SCFCOI1 and degraded via the 26S proteasome pathway.

Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1 (COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCF(COI1)) complexes with Arabidopsis thaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitination and degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemical analysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1 and further secured by assembly into SCF(COI1) complexes, suggesting a function for SCF(COI1) in the stabilization of COI1 in Arabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identified the 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated by a dynamic balance of SCF(COI1)-mediated stabilization and 26S proteasome-mediated degradation and thus maintained at a protein level essential for proper biological functions in Arabidopsis development and defense responses.

ORPA: a fast and efficient phylogenetic analysis method for constructing genome-wide alignments of organelle genomes.

Creating a multi-gene alignment matrix for phylogenetic analysis using organelle genomes involves aligning single-gene datasets manually, a process that can be time-consuming and prone to errors. The HomBlocks pipeline has been created to eliminate the inaccuracies arising from manual operations. The processing of a large number of sequences, however, remains a time-consuming task. To conquer this challenge, we develop a speedy and efficient method called Organelle Genomes for Phylogenetic Analysis (ORPA). ORPA can quickly generate multiple sequence alignments for whole-genome comparisons by parsing the result files of NCBI BLAST, completing the task just in 1 min. With increasing data volume, the efficiency of ORPA is even more pronounced, over 300 times faster than HomBlocks in aligning 60 high-plant chloroplast genomes. The phylogenetic tree outputs from ORPA are equivalent to HomBlocks, indicating its outstanding efficiency. Due to its speed and accuracy, ORPA can identify species-level evolutionary conflicts, providing valuable insights into evolutionary cognition.

Developing a multi-modular assembled prime editing (mPE) system improved precise multi-base insertion efficiency in dicots.

INTRODUCTION: The Prime Editing (PE) system is a precise and versatile genome editing tool with great potential in plant breeding and plant synthetic biology. However, low PE efficiency severely restricts its application, especially in dicots. PE can introduce small tags to trace target protein or cis-element to regulate gene transcription which is an expertise superior to other gene editing tools. Owing to low efficiency, PE adaption in stably transformed Arabidopsis is lacking. OBJECTIVES: This study aimed to investigate the issue of low PE efficiency in dicots and develop systematic solutions to improve it. Currently, PE in dicots is undetectable and inconsistent, and this study seeks to address it. Split PE into several parts showed better performance in some target sites in mammal cells. We plan to discover the optimal split PE combination in dicot. METHODS: We conducted large-scale transformation experiments in dicot model plants Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) by Agrobacterium-mediated transformation with deep amplicon sequencing (0.2-0.5 million clean total reads). RESULTS: The editing efficiency decreased upon using a fused reverse transcriptase (RT) or an extended pegRNA separately and further decreased dramatically when these were used together. With the help of the pol II strategy to express PE gRNA (pegRNA), we named the most effective split PE combination as a multi-modular assembled prime editing system (mPE). mPE exhibited improved precise editing efficiency on most gene sites with various editing types, ranging from 1.3-fold to 1288.5-fold and achieved PE on some sites that could not be edited by original PE2. Especially, mPE showed superiority for multi-base insertion with an average improvement of 197.9-fold. CONCLUSION: The original PE architecture strongly inhibited the cleavage activity of Cas9. Split PE improved PE efficiency extensively and was in favor of introducing small insertions in dicot plants, indicating that different PE variants might have their own expertise.

Mercaptoimidazole-capped gold nanoparticles as a potent agent against plant pathogenic fungi.

Plant pathogenic fungi pose a substantial challenge to agricultural production, but the conventional fungicide-based approaches are losing importance. As agents with broad-spectrum antibacterial effects, gold nanoparticles (Au NPs) are found to have antifungal effects; however, no study has examined their application in agriculture as fungicides. Accordingly, this study investigates the activity of 2-mercaptoimidazole-capped Au NPs (MI-Au NPs) against the 'top' plant pathogenic fungi, finding that they could inhibit Magnaporthe oryzae, Botrytis cinerea, Fusarium pseudograminearum and Colletotrichum destructivum by inducing cytoplasmic leakage. Moreover, MI-Au NPs are found to protect plants from infection by B. cinerea. Specifically, pot experiments demonstrate that MI-Au NPs decrease the incidence rate of B. cinerea infection in Arabidopsis thaliana from 74.6% to 6.2% and in Solanum lycopersicum from 100% to 10.9%, outperforming those achieved by imazalil. Furthermore, the biosafety assays reveal that MI-Au NPs cannot penetrate the cuticle of plant cells or negatively influence plant growth, and it is safe to mammalian cells. In summary, the findings of this study will support the development of NP-based antifungal agents for use in agriculture.

The high-quality genome of Grona styracifolia uncovers the genomic mechanism of high levels of schaftoside, a promising drug candidate for treatment of COVID-19.

Recent study has evidenced that traditional Chinese medicinal (TCM) plant-derived schaftoside shows promise as a potential drug candidate for COVID-19 treatment. However, the biosynthetic pathway of schaftoside in TCM plants remains unknown. In this study, the genome of the TCM herb Grona styracifolia (Osbeck) H.Ohashi & K.Ohashi (GSO), which is rich in schaftoside, was sequenced, and a high-quality assembly of GSO genome was obtained. Our findings revealed that GSO did not undergo recent whole genome duplication (WGD) but shared an ancestral papilionoid polyploidy event, leading to the gene expansion of chalcone synthase (CHS) and isoflavone 2'-hydroxylase (HIDH). Furthermore, GSO-specific tandem gene duplication resulted in the gene expansion of C-glucosyltransferase (CGT). Integrative analysis of the metabolome and transcriptome identified 13 CGTs and eight HIDHs involved in the biosynthetic pathway of schaftoside. Functional studies indicated that CGTs and HIDHs identified here are bona fide responsible for the biosynthesis of schaftoside in GSO, as confirmed through hairy root transgenic system and in vitro enzyme activity assay. Taken together, the ancestral papilionoid polyploidy event expanding CHSs and HIDHs, along with the GSO-specific tandem duplication of CGT, contributes, partially if not completely, to the robust biosynthesis of schaftoside in GSO. These findings provide insights into the genomic mechanisms underlying the abundant biosynthesis of schaftoside in GSO, highlighting the potential of GSO as a source of bioactive compounds for pharmaceutical development.

Heterologous synthesis of poly-γ-glutamic acid enhanced drought resistance in maize (Zea mays L.).

Drought stress is the main factor restricting maize yield. Poly-γ-glutamic acid (γ-PGA), as a water-retaining agent and fertilizer synergist, could significantly improve the drought resistance and yield of many crops. However, its high production costs and unclear long-term impact on soil ecology limit its large-scale application. In this study, an environmentally friendly green material γ-PGA was heterologous synthesized in maize for the first time using the synthetic biology method. The genes (PgsA, PgsB, PgsC) participated in γ-PGA synthesis were cloned from Bacillus licheniformis and transformed into maize to produce γ-PGA for the first time. Under drought stress, transgenic maize significantly increased the ear length, ear weight and grain weight by 50 % compared to the control, whereas the yield characteristic of ear weight, grain number per ear, grain weight per ear and 100-grain weight increased by 1.67 %-2.33 %, 3.78 %-13.06 %, 8.41 %-22.06 %, 6.03 %-19.28 %, and 11.85 %-18.36 %, respectively under normal growth conditions. γ-PGA was mainly expressed in the mesophyll cells of maize leaf rosette structure and improved drought resistance and yield by protecting and increasing the expression of genes for the photosynthetic and carbon fixation. This study is an important exploration for maize drought stress molecular breeding and building resource-saving agriculture.

Amentoflavone maintaining extracellular matrix homeostasis and inhibiting subchondral bone loss in osteoarthritis by inhibiting ERK, JNK and NF-κB signaling pathways.

Amentoflavone (AF), a plant biflavone isolated from Selaginella sinensis ethanol extract, is characterized by anti-inflammatory and anti-oxidant properties. According to previous studies, inflammation and oxidative stress are closely related to the pathophysiology of osteoarthritis (OA). However, the effects and mechanisms of AF on OA have not been elucidated.To investigate the inhibitory effects and its molecular mechanism of AF on extracellular matrix (ECM) degradation stimulated by IL-1ß as well as subchondral bone loss induced by RANKL in mice chondrocytes. Quantitative PCR was used to detect the mRNA expression of genes related to inflammation, ECM, and osteoclast differentiation. Protein expression level of iNOS, COX-2, MMP13, ADAMTS5, COL2A1, SOX9, NFATc1, c-fos, JNK, ERK, P65, IκBα was measured by western blotting. The levels of TNF-α and IL-6 in the supernatants were measured by ELISA. The amount of ECM in chondrocytes was measured using toluidine blue staining. The levels of Aggrecan and Col2a1 in chondrocytes were measured using immunofluorescence. Tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and immunofluorescence were used to detect the effect of AF on osteoclast differentiation and bone resorption. The effect of AF on destabilization of the medial meniscus (DMM)-induced OA mice can be detected in hematoxylin-eosin (H&E) staining, Safranin O green staining and immunohistochemistry.AF might drastically attenuated IL-1ß-stimulated inflammation and reduction of ECM formation by blocking ERK and NF-κB signaling pathways in chondrocytes. Meanwhile, AF suppressed the formation of osteoclasts and the resorption of bone function induced by RANKL. In vivo, AF played a protective role by stabilizing cartilage ECM and inhibiting subchondral bone loss in destabilization of the medial meniscus (DMM)-induced OA mice, further proving its protective effect in the development of OA. Our study show that AF alleviated OA by suppressing ERK, JNK and NF-κB signaling pathways in OA models in vitro and DMM-induced OA mice, suggesting that AF might be a potential therapeutic agent in the treatment of OA.

Designing a synthetic moss genome using GenoDesigner.

The de novo synthesis of genomes has made unprecedented progress and achieved milestones, particularly in bacteria and yeast. However, the process of synthesizing a multicellular plant genome has not progressed at the same pace, due to the complexity of multicellular plant genomes, technical difficulties associated with large genome size and structure, and the intricacies of gene regulation and expression in plants. Here we outline the bottom-up design principles for the de novo synthesis of the Physcomitrium patens (that is, earthmoss) genome. To facilitate international collaboration and accessibility, we have developed and launched a public online design platform called GenoDesigner. This platform offers an intuitive graphical interface enabling users to efficiently manipulate extensive genome sequences, even up to the gigabase level. This tool is poised to greatly expedite the synthesis of the P. patens genome, offering an essential reference and roadmap for the synthesis of plant genomes.

Near telomere-to-telomere genome of the model plant Physcomitrium patens.

The model plant Physcomitrium patens has played a pivotal role in enhancing our comprehension of plant evolution and development. However, the current genome harbours numerous regions that remain unfinished and erroneous. To address these issues, we generated an assembly using Oxford Nanopore reads and Hi-C mapping. The assembly incorporates telomeric and centromeric regions, thereby establishing it as a near telomere-to-telomere genome except a region in chromosome 1 that is not fully assembled due to its highly repetitive nature. This near telomere-to-telomere genome resolves the chromosome number at 26 and provides a gap-free genome assembly as well as updated gene models to aid future studies using this model organism.

Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III.

Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.