Spatial Metabolomic Profiling of Pinelliae Rhizoma from Different Leaf Types Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Pinelliae Rhizoma (PR), a highly esteemed traditional Chinese medicinal herb, is widely applied in clinical settings due to its diverse pharmacological effects, including antitussive, expectorant, antiemetic, sedative-hypnotic, and antitumor activities. Pinellia ternata exhibits morphological variation in its leaves, with types resembling peach, bamboo, and willow leaves. However, the chemical composition differences among the corresponding rhizomes of these leaf phenotypes remain unelucidated. This pioneering research employed Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) to conduct the in situ identification and spatial profiling of 35 PR metabolites in PR, comprising 12 alkaloids, 4 organic acids, 12 amino acids, 5 flavonoids, 1 sterol, and 1 anthraquinone. Our findings revealed distinct spatial distribution patterns of secondary metabolites within the rhizome tissues of varying leaf types. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) effectively differentiated between rhizomes associated with different leaf morphologies. Furthermore, this study identified five potential differential biomarkers-methylophiopogonanone B, inosine, cytidine, adenine, and leucine/isoleucine-that elucidate the biochemical distinctions among leaf types. The precise tissue-specific localization of these secondary metabolites offers compelling insights into the specialized accumulation of bioactive compounds in medicinal plants, thereby enhancing our comprehension of PR's therapeutic potential.

Spatial Mapping of Bioactive Metabolites in the Roots of Three Bupleurum Species by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Bupleurum is a kind of medicinal plant that has made a great contribution to human health because of the presence of bioactive metabolites: Bupleurum saikosaponins and flavonoids. Despite their importance, it has been a challenge to visually characterize the spatial distribution of these metabolites in situ within the plant tissue, which is essential for assessing the quality of Bupleurum. The development of a new technology to identify and evaluate the quality of medicinal plants is therefore necessary. Here, the spatial distribution and quality characteristics of metabolites of three Bupleurum species: Bupleurum smithii (BS), Bupleurum marginatum var. stenophyllum (BM), and Bupleurum chinense (BC) were characterized by Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Twenty-nine metabolites, including saikosaponins, non-saikosaponins, and compounds from the saikosaponin synthesis pathway, were characterized. Some of these were successfully localized and visualized in the transverse section of roots. In these Bupleurum species, twelve saikosaponins, five non-saikosaponins, and five saikosaponin synthesis pathway compounds were detected. Twenty-two major influencing components, which exhibit higher ion intensities in higher quality samples, were identified as potential quality markers of Bupleurum. The final outcome indicates that BC has superior quality compared to BS and BM. MALDI-MSI has effectively distinguished the quality of these Bupleurum species, providing an intuitive and effective marker for the quality control of medicinal plants.

Pm61: a recessive gene for resistance to powdery mildew in wheat landrace Xuxusanyuehuang identified by comparative genomics analysis.

KEY MESSAGE: A single recessive powdery mildew resistance gene Pm61 from wheat landrace Xuxusanyuehuang was mapped within a 0.46-cM genetic interval spanning a 1.3-Mb interval of the genomic region of chromosome arm 4AL. Epidemics of powdery mildew incited by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt) have caused significant yield reductions in many wheat (Triticum aestivum)-producing regions. Identification of powdery mildew resistance genes is required for sustainable improvement of wheat for disease resistance. Chinese wheat landrace Xuxusanyuehuang was resistant to several Bgt isolates at the seedling stage. Genetic analysis based on the inoculation of Bgt isolate E09 on the F1, F2, and F2:3 populations produced by crossing Xuxusanyuehuang to susceptible cultivar Mingxian 169 revealed that the resistance of Xuxusanyuehuang was controlled by a single recessive gene. Bulked segregant analysis and simple sequence repeat (SSR) mapping placed the gene on chromosome bin 4AL-4-0.80-1.00. Comparative genomics analysis was performed to detect the collinear genomic regions of Brachypodium distachyon, rice, sorghum, Aegilops tauschii, T. urartu, and T. turgidum ssp. dicoccoides. Based on the use of 454 contig sequences and the International Wheat Genome Sequence Consortium survey sequence of Chinese Spring wheat, four EST-SSR and seven SSR markers were linked to the gene. An F5 recombinant inbred line population derived from Xuxusanyuehuang × Mingxian 169 cross was used to develop the genetic linkage map. The gene was localized in a 0.46-cM genetic interval between Xgwm160 and Xicsx79 corresponding to 1.3-Mb interval of the genomic region in wheat genome. This is a new locus for powdery mildew resistance on chromosome arm 4AL and is designated Pm61.

Quantitative Phosphoproteomic Analysis Provides Insight into the Response to Short-Term Drought Stress in Ammopiptanthus mongolicus Roots.

Drought is one of the major abiotic stresses that negatively affects plant growth and development. Ammopiptanthus mongolicus is an ecologically important shrub in the mid-Asia desert region and used as a model for abiotic tolerance research in trees. Protein phosphorylation participates in the regulation of various biological processes, however, phosphorylation events associated with drought stress signaling and response in plants is still limited. Here, we conducted a quantitative phosphoproteomic analysis of the response of A. mongolicus roots to short-term drought stress. Data are available via the iProx database with project ID IPX0000971000. In total, 7841 phosphorylation sites were found from the 2019 identified phosphopeptides, corresponding to 1060 phosphoproteins. Drought stress results in significant changes in the abundance of 103 phosphopeptides, corresponding to 90 differentially-phosphorylated phosphoproteins (DPPs). Motif-x analysis identified two motifs, including [pSP] and [RXXpS], from these DPPs. Functional enrichment and protein-protein interaction analysis showed that the DPPs were mainly involved in signal transduction and transcriptional regulation, osmotic adjustment, stress response and defense, RNA splicing and transport, protein synthesis, folding and degradation, and epigenetic regulation. These drought-corresponsive phosphoproteins, and the related signaling and metabolic pathways probably play important roles in drought stress signaling and response in A. mongolicus roots. Our results provide new information for understanding the molecular mechanism of the abiotic stress response in plants at the posttranslational level.

PmLX66 and PmW14: New Alleles of Pm2 for Resistance to Powdery Mildew in the Chinese Winter Wheat Cultivars Liangxing 66 and Wennong 14.

Wheat powdery mildew (caused by Blumeria graminis f. sp. tritici) can be effectively managed by growing resistant cultivars. 'Liangxing 66' and 'Wennong 14' are the current winter wheat cultivars grown in northern China where powdery mildew is epidemic. Both cultivars have been demonstrated to carry single dominant genes for resistance to powdery mildew, tentatively designated PmLX66 and PmW14, on chromosome 5DS and share common linked markers with Pm2. Allelism tests were performed using a total of 15,657 plants of F2 segregating populations to determine the relationship between PmLX66, PmW14, and Pm2. All progeny from the crosses Liangxing 66 × 'Ulka/8*Chancellor' (Ulka/8*Cc), Wennong 14 × Ulka/8*Cc, and Liangxing 66 × Wennong 14 were resistant when tested with B. graminis f. sp. tritici isolate E20, indicating that PmLX66 and PmW14 are allelic to Pm2 and to each other. Liangxing 66 was resistant to 76.7% of the 60 B. graminis f. sp. tritici isolates from northern China, a slightly smaller proportion than Ulka/8*Cc (78.3%). However, Wennong 14 (85.0%) was more resistant against this set of B. graminis f. sp. tritici isolates than Ulka/8*Cc and Liangxing 66. Liangxing 66 and Wennong 14 differed from Ulka/8*Cc in respect to a number of B. graminis f. sp. tritici isolates. Based on these findings, PmLX66 and PmW14 are new alleles at the Pm2 locus.

Effects of elicitors from culture filtrate of Fusarium solani CL105 on flavonoid production of Scutellaria baicalensis calli.

Introduction: Endophytic fungi can promote secondary metabolite accumulation in medicinal plants. Previously, we observed that the culture filtrate of Fusarium solani CL105 promoted flavonoid production in Scutellaria baicalensis calli. However, the active ingredients and mechanisms associated with this secondary metabolite accumulation remain unclear. Methods: This study evaluates the effects of different elicitors from the culture filtrate of F. solani CL105 namely, exopolysaccharide (EPS), exoprotein (EP), and other parts (OP), on the flavonoid production in S. baicalensis calli by HPLC. Subsequently, the underlying mechanism of EPS induced flavonoid production in S. baicalensis calli was revealed by transcriptomics and RT-PCR. Results and discussion: The results indicated a significant increase in flavonoid production in S. baicalensis calli following treatment with EPS. Baicalin (1.40 fold), wogonoside (1.91 fold), and wogonin (2.76 fold) were most significantly up-regulated compared with the control. Transcriptome analysis further revealed up-regulation of key enzyme genes (CHS, CHI, FNS, and F6H) involved in flavonoid synthesis after 5 days of EPS treatment. Moreover, the expression of GA2ox and CYP707A-genes involved in gibberellin acid (GA) and abscisic acid biosynthesis (ABA), respectively-were significantly up-regulated. The expression levels of certain transcription factors, including MYB3, MYB8, and MYB13, were also significantly higher than in controls. Our results indicated that EPS was a main active elicitor involved in promoting flavonoid production in S. baicalensis calli. We postulated that EPS might stimulate the expression of MYB3, MYB8, MYB13, GA2ox, and CYP707A, leading to markedly upregulated CHS, CHI, FNS, and F6H expression levels, ultimately promoting flavonoid synthesis. This study provides a novel avenue for large-scale in vitro production of flavonoids in S. baicalensis.

Spatial distribution of metabolites in processing Ziziphi Spinosae Semen as revealed by matrix-assisted laser desorption/ionization mass spectrometry imaging.

Ziziphi Spinosae Semen (ZSS) is the first choice for the treatment of insomnia. This research aimed to reveal the spatial distribution of identifying quality markers of ZSS and to illustrate the metabolite quality characteristics of this herbal medicine. Here, we performed a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in situ to detect and image 33 metabolites in ZSS, including three saponins, six flavonoids, four alkaloids, eight fatty acids, and 12 amino acids. The MALDI images of the metabolites clearly showed the heterogeneous spatial distribution in different regions of ZSS tissues, such as the cotyledon, endosperm, and radicle. The distribution area of two saponins, six flavonoids, and three alkaloids increased significantly after the fried processing of ZSS. Based on the ion images, samples with different processing technologies were distinguished unambiguously by the pattern recognition method of orthogonal partial least squares discrimination analysis (OPLS-DA). Simultaneously, 23 major influencing components exerting higher ion intensities were identified as the potential quality markers of ZSS. Results obtained in the current research demonstrate that the processing of ZSS changes its content and distribution of the medicinal components. The analysis of MALDI-MSI provides a novel MS-based molecular imaging approach to investigate and monitor traditional medicinal plants.

Genetic analysis and detection of the gene MlLX99 on chromosome 2BL conferring resistance to powdery mildew in the wheat cultivar Liangxing 99.

KEY MESSAGE: The effectiveness of wheat cultivar Liangxing 99 against powdery mildew was shown to be controlled by a single dominant gene located on a new locus of chromosome 2BL in the bin 2BL2-0.35-0.50. Liangxing 99, one of the most widely grown commercial cultivars in the winter wheat (Triticum aestivum) producing regions in northern China, was shown to provide a broad spectrum of resistance to Blumeria graminis f. sp. tritici (Bgt) isolates originating from that region. Using an F2 population and F2:3 lines derived from a cross of Liangxing 99 × Zhongzuo 9504, genetic analysis demonstrated that a single dominant gene, designated MlLX99, was responsible for the resistance of Liangxing 99 to Bgt isolate E09. The results of molecular analysis indicated that this gene is located on chromosome 2BL and flanked by the SSR marker Xgwm120 and EST-STS marker BE604758 at genetic distances of 2.9 and 5.5 cM, respectively. Since the flanking markers of MlLX99 were previously mapped to the bin 2BL2-0.36-0.50, MlLX99 must be located in this chromosomal region. MlLX99 showed a different resistance reaction pattern to 60 Bgt isolates from Pm6, Pm33, and PmJM22, which were all previously mapped on chromosome 2BL, but differed in their positions from MlLX99. Due to its unique position on chromosome 2BL, MlLX99 appears to be a new locus for resistance to powdery mildew. Liangxing 99 has shown superior yield performance and wide adaptation to different agricultural conditions, which has resulted in its extensive use as a wheat cultivar in China. The identification of resistance gene MlLX99 facilitates the use of this cultivar in the protection of wheat from damage caused by powdery mildew.

DNA barcoding identification of grafted Semen Ziziphi Spinosae and transcriptome study of wild Semen Ziziphi Spinosae.

Semen Ziziphi Spinosae (SZS) is the dried and ripe seeds of Ziziphus jujuba var. spinosa. Currently, the yield of naturally grown SZS is unstable owing to environmental factors. Grafting high-quality sour jujube scions onto sour jujube or jujube tree stocks can result in a greater yield. However, the effects of grafting on the quality and gene expression of SZS have rarely been reported. This study used a DNA barcoding technique, high-performance liquid phase-evaporative luminescence detector (HPLC-ELSD), and transcriptomics to investigate the origin and genetic differences between grafted and wild jujube seeds. DNA barcoding identified all samples as Ziziphus jujuba var. spinosa. HPLC-ELSD analysis revealed a higher content of grafted SZS compared to that of the wild SZS. Transcriptome analysis of the metabolic pathways in SZS showed that 22 and 19 differentially expressed gene sequences encoded enzymes related to flavonoids and saponin synthesis, respectively. Weighted correlation network analysis (WGCNA) identified 15 core genes governing the differences in medicinal components between grafted and wild SZS. This study demonstrated the use of DNA barcoding and fingerprint methods to identify jujube seed species and effectively capture ingredient information of medicinal materials. Additionally, transcriptome technology provided data for identifying core differential genes, facilitating studies on quality differences between grafted and wild SZS.

The Characterization of R2R3-MYB Genes in Ammopiptanthus nanus Uncovers That the miR858-AnaMYB87 Module Mediates the Accumulation of Anthocyanin under Osmotic Stress.

R2R3-MYB transcription factors (TFs) participate in the modulation of plant development, secondary metabolism, and responses to environmental stresses. Ammopiptanthus nanus, a leguminous dryland shrub, tolerates a high degree of environmental stress, including drought and low-temperature stress. The systematic identification, structural analysis, evolutionary analysis, and gene profiling of R2R3-MYB TFs under cold and osmotic stress in A. nanus were performed. Up to 137 R2R3-MYB TFs were identified and clustered into nine clades, with most A. nanus R2R3-MYB members belonging to clade VIII. Tandem and segmental duplication events drove the expansion of the A. nanus R2R3-MYB family. Expression profiling revealed that multiple R2R3-MYB genes significantly changed under osmotic and cold stress conditions. MiR858 and miR159 targeted 88 R2R3-MYB genes. AnaMYB87, an miR858-targeted clade VIII R2R3-MYB TF, was up-regulated under both osmotic and cold stress. A transient expression assay in apples showed that the overexpression of AnaMYB87 promoted anthocyanin accumulation. A luciferase reporter assay in tobacco demonstrated that AnaMYB87 positively affected the transactivation of the dihydroflavonol reductase gene, indicating that the miR858-MYB87 module mediates anthocyanin accumulation under osmotic stress by regulating the dihydroflavonol reductase gene in A. nanus. This study provides new data to understand the roles of R2R3-MYB in plant stress responses.

The complete chloroplast genome of Piptanthus nepalensis, a medicinal plant.

Piptanthus nepalensis (Hooker) Sweet is a semi deciduous or deciduous shrub belonging to the genus Piptanthus, Leguminosae. P. nepalensis has been used as a folk medicinal herb in Nepal and was cultivated all over the world as an ornamental plant. In the present study, we sequenced the entire genome of the chloroplast of P. nepalensis. The total length of the chloroplast genome in P. nepalensis is 152,195 bp, including a large single-copy region of 82,048 bp, a small single-copy region of 17,675 bp, and a pair of inverted repeats regions of 26,236 bp. The overall guanine-cytosine (GC) content of the genome was 36.7%. There are 131 genes in the chloroplast genome of P. nepalensis, including 85 protein-coding genes, 8 rRNA genes and 38 tRNA genes. Phylogenetic analysis showed that P. nepalensis is closely related to Maackia floribunda.

The complete chloroplast genome of Sophora davidii (Fabaceae) and its phylogenetic implications.

Sophora davidii (Franch.) Pavol. is a deciduous or evergreen shrubs belonging to the genus Sophora, Fabaceae. The roots of S. davidii have been traditionally used as a medicinal herb in China to clear internal heat, relieve sore throat, and reduce swelling. Here we sequenced the whole genome of the chloroplast of S. davidii. The complete length of the chloroplast genome in S. davidii is 153,584 bp, containing a large single-copy region of 83,930 bp, a small single-copy region of 15,008 bp, and a pair of inverted repeats regions of 25,823 bp. The total guanine-cytosine (GC) percentage of the chloroplast genome was 36.7%. A total of 131 genes were annotated from the chloroplast genome of S. davidii, including 85 protein-coding genes, 8 rRNA genes and 38 tRNA genes. The phylogenetic analysis showed that S. davidii is closely related to the other three species of genus Sophora.

Identification of miRNAs and Their Response to Cold Stress in Astragalus Membranaceus.

Astragalus membranaceus is an important medicinal plant widely cultivated in East Asia. MicroRNAs (miRNAs) are endogenous regulatory molecules that play essential roles in plant growth, development, and the response to environmental stresses. Cold is one of the key environmental factors affecting the yield and quality of A. membranaceus, and miRNAs may mediate the gene regulation network under cold stress in A. membranaceus. To identify miRNAs and reveal their functions in cold stress response in A. membranaceus, small RNA sequencing was conducted followed by bioinformatics analysis, and quantitative real time PCR (qRT-PCR) analysis was performed to profile the expression of miRNAs under cold stress. A total of 168 conserved miRNAs belonging to 34 families and 14 putative non-conserved miRNAs were identified. Many miRNA targets were predicted and these targets were involved in diversified regulatory and metabolic pathways. By using qRT-PCR, 27 miRNAs were found to be responsive to cold stress, including 4 cold stress-induced and 17 cold-repressed conserved miRNAs, and 6 cold-induced non-conserved miRNAs. These cold-responsive miRNAs probably mediate the response to cold stress by regulating development, hormone signaling, defense, redox homeostasis, and secondary metabolism in A. membranaceus. These cold-corresponsive miRNAs may be used as the candidate genes in further molecular breeding for improving cold tolerance of A. membranaceus.

Long-read sequencing and de novo genome assembly of Ammopiptanthus nanus, a desert shrub.

Background: Ammopiptanthus nanus is a rare broad-leaved shrub that is found in the desert and arid regions of Central Asia. This plant species exhibits extremely high tolerance to drought and freezing and has been used in abiotic tolerance research in plants. As a relic of the tertiary period, A. nanus is of great significance to plant biogeographic research in the ancient Mediterranean region. Here, we report a draft genome assembly using the Pacific Biosciences (PacBio) platform and gene annotation for A. nanus. Findings: A total of 64.72 Gb of raw PacBio sequel reads were generated from four 20-kb libraries. After filtering, 64.53 Gb of clean reads were obtained, giving 72.59× coverage depth. Assembly using Canu gave an assembly length of 823.74 Mb, with a contig N50 of 2.76 Mb. The final size of the assembled A. nanus genome was close to the 889 Mb estimated by k-mer analysis. The gene annotation completeness was evaluated using Benchmarking Universal Single-Copy Orthologs; 1,327 of the 1,440 conserved genes (92.15%) could be found in the A. nanus assembly. Genome annotation revealed that 74.08% of the A. nanus genome is composed of repetitive elements and 53.44% is composed of long terminal repeat elements. We predicted 37,188 protein-coding genes, of which 96.53% were functionally annotated. Conclusions: The genomic sequences of A. nanus could be a valuable source for comparative genomic analysis in the legume family and will be useful for understanding the phylogenetic relationships of the Thermopsideae and the evolutionary response of plant species to the Qinghai Tibetan Plateau uplift.