Knockout of CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L enhances the S/G ratio of lignin monomers and disease resistance in Nicotiana tabacum.

Background: Nicotiana tabacum is an important economic crop, which is widely planted in the world. Lignin is very important for maintaining the physiological and stress-resistant functions of tobacco. However, higher lignin content will produce lignin gas, which is not conducive to the formation of tobacco quality. To date, how to precisely fine-tune lignin content or composition remains unclear. Results: Here, we annotated and screened 14 CCoAOMTs in Nicotiana tabacum and obtained homozygous double mutants of CCoAOMT6 and CCoAOMT6L through CRSIPR/Cas9 technology. The phenotype showed that the double mutants have better growth than the wild type whereas the S/G ratio increased and the total sugar decreased. Resistance against the pathogen test and the extract inhibition test showed that the transgenic tobacco has stronger resistance to tobacco bacterial wilt and brown spot disease, which are infected by Ralstonia solanacearum and Alternaria alternata, respectively. The combined analysis of metabolome and transcriptome in the leaves and roots suggested that the changes of phenylpropane and terpene metabolism are mainly responsible for these phenotypes. Furthermore, the molecular docking indicated that the upregulated metabolites, such as soyasaponin Bb, improve the disease resistance due to highly stable binding with tyrosyl-tRNA synthetase targets in Ralstonia solanacearum and Alternaria alternata. Conclusions: CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L can regulate the S/G ratio of lignin monomers and may affect tobacco bacterial wilt and brown spot disease resistance by disturbing phenylpropane and terpene metabolism in leaves and roots of Nicotiana tabacum, such as soyasaponin Bb.

Evaluation of Cooked Rice for Eating Quality and Its Components in Geng Rice.

At present, ''eating well" is increasingly desired by people instead of merely ''being full". Rice provides the majority of daily caloric needs for half of the global human population. However, eating quality is difficult to objectively evaluate in rice breeding programs. This study was carried out to objectively quantify and predict eating quality in Geng rice. First, eating quality and its components were identified by trained panels. Analysis of variance and broad-sense heritability showed that variation among varieties was significant for all traits except hardness. Among them, viscosity, taste, and appearance were significantly correlated with eating quality. We established an image acquisition and processing system to quantify cooked rice appearance and optimized the process of measuring cooked rice viscosity with a texture analyzer. The results show that yellow areas of the images were significantly correlated with appearance, and adhesiveness was significantly correlated with viscosity. Based on these results, multiple regression analysis was used to predict eating quality: eating quality = 0.37 × adhesiveness - 0.71 × yellow area + 0.89 × taste - 0.34, R2 = 0.85. The correlation coefficient between the predicted and actual values was 0.86. We anticipate that this predictive model will be useful in future breeding programs for high-eating-quality rice.

Biosynthesis and transport of pollen coat precursors in angiosperms.

The pollen coat is a hydrophobic mixture on the pollen grain surface, which plays an important role in protecting male gametes from various environmental stresses and microorganism attacks, and in pollen-stigma interactions during pollination in angiosperms. An abnormal pollen coat can result in humidity-sensitive genic male sterility (HGMS), which can be used in two-line hybrid crop breeding. Despite the crucial functions of the pollen coat and the application prospect of its mutants, few studies have focused on pollen coat formation. In this Review, the morphology, composition and function of different types of pollen coat are assessed. On the basis of the ultrastructure and development process of the anther wall and exine found in rice and Arabidopsis, the genes and proteins involved in the biosynthesis of pollen coat precursors and the possible transport and regulation process are sorted. Additionally, current challenges and future perspectives, including potential strategies utilizing HGMS genes in heterosis and plant molecular breeding, are highlighted.

The global integrative network: integration of signaling and metabolic pathways.

The crosstalk between signaling and metabolic pathways has been known to play key roles in human diseases and plant biological processes. The integration of signaling and metabolic pathways can provide an essential reference framework for crosstalk analysis. However, current databases use distinct structures to present signaling and metabolic pathways, which leads to the chaos in the integrated networks. Moreover, for the metabolic pathways, the metabolic enzymes and the reactions are disconnected by the current widely accepted layout of edges and nodes, which hinders the topological analysis of the integrated networks. Here, we propose a novel "meta-pathway" structure, which uses the uniformed structure to display the signaling and metabolic pathways, and resolves the difficulty in linking the metabolic enzymes to the reactions topologically. We compiled a comprehensive collection of global integrative networks (GINs) by merging the meta-pathways of 7077 species. We demonstrated the assembly of the signaling and metabolic pathways using the GINs of four species-human, mouse, Arabidopsis, and rice. Almost all of the nodes were assembled into one major network for each of the four species, which provided opportunities for robust crosstalk and topological analysis, and knowledge graph construction. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-022-00078-1.

The genome of Dioscorea zingiberensis sheds light on the biosynthesis, origin and evolution of the medicinally important diosgenin saponins.

Diosgenin saponins isolated from Dioscorea species such as D. zingiberensis exhibit a broad spectrum of pharmacological activities. Diosgenin, the aglycone of diosgenin saponins, is an important starting material for the production of steroidal drugs. However, how plants produce diosgenin saponins and the origin and evolution of the diosgenin saponin biosynthetic pathway remain a mystery. Here we report a high-quality, 629-Mb genome of D. zingiberensis anchored on 10 chromosomes with 30 322 protein-coding genes. We reveal that diosgenin is synthesized in leaves ('source'), then converted into diosgenin saponins, and finally transported to rhizomes ('sink') for storage in plants. By evaluating the distribution and evolutionary patterns of diosgenin saponins in Dioscorea species, we find that diosgenin saponin-containing may be an ancestral trait in Dioscorea and is selectively retained. The results of comparative genomic analysis indicate that tandem duplication coupled with a whole-genome duplication event provided key evolutionary resources for the diosgenin saponin biosynthetic pathway in the D. zingiberensis genome. Furthermore, comparative transcriptome and metabolite analysis among 13 Dioscorea species suggests that specific gene expression patterns of pathway genes promote the differential evolution of the diosgenin saponin biosynthetic pathway in Dioscorea species. Our study provides important insights and valuable resources for further understanding the biosynthesis, evolution, and utilization of plant specialized metabolites such as diosgenin saponins.

Improving bread wheat yield through modulating an unselected AP2/ERF gene.

Crop breeding heavily relies on natural genetic variation. However, additional new variations are desired to meet the increasing human demand. Inflorescence architecture determines grain number per spike, a major determinant of bread wheat (Triticum aestivum L.) yield. Here, using Brachypodium distachyon as a wheat proxy, we identified DUO-B1, encoding an APETALA2/ethylene response factor (AP2/ERF) transcription factor, regulating spike inflorescence architecture in bread wheat. Mutations of DUO-B1 lead to mild supernumerary spikelets, increased grain number per spike and, importantly, increased yield under field conditions without affecting other major agronomic traits. DUO-B1 suppresses cell division and promotes the expression of BHt/WFZP, whose mutations could lead to branched 'miracle-wheat'. Pan-genome analysis indicated that DUO-B1 has not been utilized in breeding, and holds promise to increase wheat yield further.

Jujube metabolome selection determined the edible properties acquired during domestication.

Plants supply both food and medicinal compounds, which are ascribed to diverse metabolites produced by plants. However, studies on domestication-driven changes in the metabolome and genetic basis of bioactive molecules in perennial fruit trees are generally lacking. Here, we conducted multidimensional analyses revealing a singular domestication event involving the genomic and metabolomic selection of jujube trees (Ziziphus jujuba Mill.). The genomic selection for domesticated genes was highly enriched in metabolic pathways, including carbohydrates and specialized metabolism. Domesticated metabolome profiling indicated that 187 metabolites exhibited significant divergence as a result of directional selection. Malic acid was directly selected during domestication, and the simultaneous selection of specialized metabolites, including triterpenes, consequently lead to edible properties. Cyclopeptide alkaloids (CPAs) were specifically targeted for the divergence between dry and fresh cultivars. We identified 1080 significantly associated loci for 986 metabolites. Among them, 15 triterpenes were directly selected at six major loci, allowing the identification of a homologous cluster containing seven 2,3-oxidosqualene cyclases (OSCs). An OSC gene was found to contribute to the reduction in the content of triterpenes during domestication. The complete pathway for synthesizing ursolic acid was dissected by integration of the metabolome and transcriptome. Additionally, an N-methyltransferase involved in the biosynthesis of CPA and responsible for inter-cultivar content variation was identified. The present study promotes our understanding of the selection process of the global metabolome subsequent to fruit tree domestication and facilitates the genetic manipulation of specialized metabolites to enhance their edible traits.

Diverse triterpene skeletons are derived from the expansion and divergent evolution of 2,3-oxidosqualene cyclases in plants.

Triterpenoids are one of the largest groups of secondary metabolites and exhibit diverse structures, which are derived from C30 skeletons that are biosynthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene. Triterpenoids have a wide range of biological activities, and are used in functional foods, drugs, and as industrial materials. Due to the low content levels in their native plants and limited feasibility and efficiency of chemical synthesis, heterologous biosynthesis of triterpenoids is the most promising strategy. Herein, we classified 121 triterpene alcohols/ketones according to their conformation and ring numbers, among which 51 skeletons have been experimentally characterized as the products of oxidosqualene cyclases (OSCs). Interestingly, 24 skeletons that have not been reported from nature source were generated by OSCs in heterologous expression. Comprehensive evolutionary analysis of the identified 152 OSCs from 75 species in 25 plant orders show that several pentacyclic triterpene synthases repeatedly originated in multiple plant lineages. Comparative analysis of OSC catalytic reaction revealed that stabilization of intermediate cations, steric hindrance, and conformation of active center amino acid residues are primary factors affecting triterpene formation. Optimization of OSC could be achieved by changing of side-chain orientations of key residues. Recently, methods, such as rationally design of pathways, regulation of metabolic flow, compartmentalization engineering, etc., were introduced in improving chassis for the biosynthesis of triterpenoids. We expect that extensive study of natural variation of large number of OSCs and catalytical mechanism will provide basis for production of high level of triterpenoids by application of synthetic biology strategies.

Mining plant metabolomes: Methods, applications, and perspectives.

Plants produce a variety of metabolites that are essential for plant growth and human health. To fully understand the diversity of metabolites in certain plants, lots of methods have been developed for metabolites detection and data processing. In the data-processing procedure, how to effectively reduce false-positive peaks, analyze large-scale metabolic data, and annotate plant metabolites remains challenging. In this review, we introduce and discuss some prominent methods that could be exploited to solve these problems, including a five-step filtering method for reducing false-positive signals in LC-MS analysis, QPMASS for analyzing ultra-large GC-MS data, and MetDNA for annotating metabolites. The main applications of plant metabolomics in species discrimination, metabolic pathway dissection, population genetic studies, and some other aspects are also highlighted. To further promote the development of plant metabolomics, more effective and integrated methods/platforms for metabolite detection and comprehensive databases for metabolite identification are highly needed. With the improvement of these technologies and the development of genomics and transcriptomics, plant metabolomics will be widely used in many fields.

Mapping causal genes and genetic interactions for agronomic traits using a large F2 population in rice.

Dissecting the genetic mechanisms underlying agronomic traits is of great importance for crop breeding. Agronomic traits are usually controlled by multiple quantitative trait loci (QTLs) and genetic interactions, and mapping the underlying causal genes is still labor-intensive and time-consuming. Here, we present a genetic tool for directly targeting the specific causal genes by using a single-gene resolution linkage map that was constructed from 3756 F2 rice plants via targeted sequencing technology and Tukey-Kramer multiple comparisons test. Three large- and moderate-effect QTLs, qHD6-2, qGL3-1, and qGW5-2, were successfully mapped to their specific causal genes, Hd1, GS3, and GW5, respectively. A complex genetic interaction network containing 30 QTL-QTL interactions was constructed, revealing that the alternative allele of hub QTL, qHD6-2, can hide or release the genetic contributions of the alleles at interacting loci. Moreover, arranging genetic interactions in the models lead to more accurate phenotypic predictions. These results provide a community resource and new feasible strategy for deciphering the genetic mechanisms of complex agronomic traits and accelerating crop breeding.

Expansion within the CYP71D subfamily drives the heterocyclization of tanshinones synthesis in Salvia miltiorrhiza.

Tanshinones are the bioactive nor-diterpenoid constituents of the Chinese medicinal herb Danshen (Salvia miltiorrhiza). These groups of chemicals have the characteristic furan D-ring, which differentiates them from the phenolic abietane-type diterpenoids frequently found in the Lamiaceae family. However, how the 14,16-epoxy is formed has not been elucidated. Here, we report an improved genome assembly of Danshen using a highly homozygous genotype. We identify a cytochrome P450 (CYP71D) tandem gene array through gene expansion analysis. We show that CYP71D373 and CYP71D375 catalyze hydroxylation at carbon-16 (C16) and 14,16-ether (hetero)cyclization to form the D-ring, whereas CYP71D411 catalyzes upstream hydroxylation at C20. In addition, we discover a large biosynthetic gene cluster associated with tanshinone production. Collinearity analysis indicates a more specific origin of tanshinones in Salvia genus. It illustrates the evolutionary origin of abietane-type diterpenoids and those with a furan D-ring in Lamiaceae.

Characterization of a 2,3-oxidosqualene cyclase in the toosendanin biosynthetic pathway of Melia toosendan.

Toosendanin, bearing a furan ring, is a limonoid belonging to the group of tetranortriterpenoids. Toosendanin is a phytochemical found in the medicinal plant Melia toosendan Sieb. et Zucc. of the Meliaceae family. Toosendanin and its derivatives demonstrate high insecticidal activity and are important pesticides derived from plants. Despite intensive investigation of limonoids over several decades, the biosynthetic pathway of these triterpenoids is less understood. To identify the key enzymes involved in the toosendanin biosynthetic pathway, we analyzed the contents of toosendanin in various plant tissues and parts and found that the highest level of toosendanin was found in the developing fruit and gradually decreased as the fruit matured. More than 346 116 transcripts were assembled based on 394 million paired-end Illumina reads and 6 million PacBio reads from the pooled RNA samples of fruits, leaves and young barks. A total of 186 263 genes were predicted. Six 2,3-oxidosqualene cyclase (OSC) genes were identified by analyzing the association between gene expression and metabolite profiles. Functional analyses using the Nicotiana benthamiana transient expression assay showed that MtOSC1 catalyzed 2,3-oxidosqualene to produce a tetracyclic triterpene skeleton, tirucalla-7,24-dien-3ß-ol, which is predicted as the precursor for toosendanin biosynthesis. We identified another OSC, MtOSC6, which is a lupeol synthase. Using synthetic biology methods, these identified enzymes could be used to model a biosynthetic pathway to produce large quantities of toosendanin.

QPMASS: A parallel peak alignment and quantification software for the analysis of large-scale gas chromatography-mass spectrometry (GC-MS)-based metabolomics datasets.

Gas chromatography-mass spectrometry (GC-MS) is a robust analytical platform for analysis of small molecules. Recently, it is widely used for large-scale metabolomics studies, in which hundreds or even thousands of samples are analyzed simultaneously, producing a very large and complex GC-MS datasets. A number of software are currently available for processing GC-MS data, but it is too compute-intensive for them to efficiently and accurately align chromatographic peaks from thousands of samples. Here, we report a newly developed software, QPMASS, for analysis of large-scale GC-MS data. The parallel computing with an advanced dynamic programming approach is implemented in QPMASS to align peaks from multiple samples based on retention time and mass spectra, enabling fast processing large-scale datasets. Furthermore, the missing value filtering and backfilling are introduced into the program, greatly reducing false positive and false negative errors to be less than 5%. We demonstrated that it took only 8 h to align and quantify a GC-TOF-MS dataset from 300 rice leaves samples, and 17 h to process a GC-qMS dataset from 1000 rice seed samples by using a personal computer (3.70 GHz CPU, 16 GB of memory and > 100 GB hard disk). QPMASS is written in C++ programming language, and is able to run under Windows operation system with a user-friendly interface.

Cytochrome P450 family member CYP96B5 hydroxylates alkanes to primary alcohols and is involved in rice leaf cuticular wax synthesis.

Odd-numbered primary alcohols are components of plant cuticular wax, but their biosynthesis remains unknown. We isolated a rice wax crystal-sparse leaf 5 (WSL5) gene using a map-based cloning strategy. The function of WSL5 was illustrated by overexpression and knockout in rice, heterologous expression in Arabidopsis and transient expression in tobacco leaves. WSL5 is predicted to encode a cytochrome P450 family member CYP96B5. The wsl5 mutant lacked crystalloid platelets on the surface of cuticle membrane, and its cuticle membrane was thicker than that of the wild-type. The wsl5 mutant is more tolerant to drought stress. The load of C23 -C33 alkanes increased, whereas the C29 primary alcohol reduced significantly in wsl5 mutant and WSL5 knockout transgenic plants. Overexpression of WSL5 increased the C29 primary alcohol and decreased alkanes in rice leaves. Heterologous expression of WSL5 increased the C29 primary alcohol and decreased alkanes, secondary alcohol, and ketone in Arabidopsis stem wax. Transient expression of WSL5 in tobacco leaves also increased the production C29 primary alcohol. WSL5 catalyzes the terminal hydroxylation of alkanes, yielding odd-numbered primary alcohols, and is involved in the formation of epidermal wax crystals on rice leaf, affecting drought sensitivity.

Orthologous receptor kinases quantitatively affect the host status of barley to leaf rust fungi.

Global food security depends on cereal crops with durable disease resistance. Most cereals are colonized by rust fungi, which are pathogens of major significance for global agriculture1. Cereal rusts display a high degree of host specificity and one rust species or forma specialis generally colonizes only one cereal host2. Exploiting the non-host status and transferring non-host resistance genes between cereal crop species has been proposed as a strategy for durable rust resistance breeding. The molecular determinants that define the host status to rusts, however, are largely unknown. Here, we show that orthologous genes at the Rphq2 locus for quantitative leaf rust resistance from cultivated barley3 and Rph22 from wild bulbous barley4 affect the host status to leaf rusts. Both genes encode lectin receptor-like kinases. We transformed Rphq2 and Rph22 into an experimental barley line that has been bred for susceptibility to non-adapted leaf rusts, which allowed us to quantify resistance responses against various leaf rust species. Rphq2 conferred a much stronger resistance to the leaf rust of wild bulbous barley than to the leaf rust adapted to cultivated barley, while for Rph22 the reverse was observed. We hypothesize that adapted leaf rust species mitigate perception by cognate host receptors by lowering ligand recognition. Our results provide an example of orthologous genes that connect the quantitative host with non-host resistance to cereal rusts. Such genes provide a basis to exploit non-host resistance in molecular breeding.

Improvement of multiplex semi-nested PCR system for screening of rare mutations by high-throughput sequencing.

The CRISPR/Cas9 system is an efficient gene-editing method, but it is difficult to obtain mutants for some specific species and special genome structures. A previously reported multiplexed, semi-nested PCR target-enrichment approach, which does not rely on transgenic technology, has been shown to be an effective and affordable strategy for the discovery of rare mutations in a large sodium azide-induced rice population. However, this strategy has the potential for further optimization. Here, we describe an improved multiplex semi-nested PCR target-enrichment strategy with simplified processing procedures, reduced false-positive rates and increased mutation detection frequency (1 mutation/73 Kb).

An Efficient System for Ds Transposon Tagging in Brachypodium distachyon.

Transposon tagging is a powerful tool that has been widely applied in several species for insertional mutagenesis in plants. Several efforts have aimed to create transfer-DNA (T-DNA) insertional mutant populations in Brachypodium distachyon, a monocot plant used as a model system to study temperate cereals, but there has been a lack of research aimed at using transposon strategies. Here, we describe the application of a maize (Zea mays) Dissociation (Ds) transposon tagging system in B distachyon The 35S::AcTPase cassette and Ds element were constructed within the same T-DNA and transformed into B distachyon plants. The Ds element was readily transposed to other chromosomes or to the same chromosome under the function of Activator (Ac) transposase. Through homologous chromosome synapsis, recombination, and segregation, the Ds element separated from the Ac element. We selected stable Ds-only plants using G418 and GFP assays and analyzed 241 T0 lines, some of which were highly efficient at producing Ds-only progeny. Through thermal asymmetric interlaced PCR, we isolated 710 independent Ds flanking sequences from Ds-only plants. Furthermore, we identified a large collection of mutants with visible developmental phenotypes via this transposon tagging system. The system is relatively simple and rapid in comparison to traditional T-DNA insertion strategies, because once efficiency lines are obtained they can be reused to generate more lines from nontransposed plants without the use of time-consuming tissue culture steps.