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Key Message: NnNAC100-NnSBEII modules enhance starch content of the rhizome in Nelumbo nucifera Gaertn. Nelumbo nucifera Gaertn. is a popular aquatic vegetable and traditional Chinese medicine whose quality and taste are mainly determined by the starch. Although starch-related genes have been functionally characterized, the regulated mechanism of enzyme (SBE) remains unclear. In this study, we identified and functionally elucidated the functions of NnSBEII and NnNAC100 using transient overexpression of NnSBEII and NnNAC100 in rhizomes of lotus, and it significantly increased the amylopectin content and total starch content. Accordingly, functional complementation assay in defective Arabidopsis also showed that NnSBEII compensated for the low content of starch in the mutant sbe2.2. In addition, overexpression of NnSBEII and NnNAC100 significantly increased the content of starch in transgenic lines. Consistently, opposite results were observed under the background of repressed NnSBEII and NnNAC100 in rhizomes of lotus. Furthermore, yeast one-hybrid and dual-luciferase assays revealed that NnNAC100 could directly bind to the NnSBEII promoter and promote the expression of NnSBEII. Transient overexpression of NnNAC100 upregulated NnSBEII expression significantly, while the expression level of AtSBE2.2 in transgenic Arabidopsis overexpressing NnNAC100 was higher than that of WT, which indicated that NnNAC100 promoted the synthesis of amylopectin by enhancing the expression of NnSBEII. In addition, we found that NnSBEII could form a complex protein by interacting with soluble starch synthase (NnSS2) to increase the activity of the SBEII enzyme. These results reveal a novel mechanism that the NnNAC100-NnSBEII-NnSBEII/NnSS2 module regulates amylopectin biosynthesis and these will provide insights into the broader implications of the regulation mechanism of starch biosynthesis., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no conflict of interest., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Sanqi, the root of Panax notoginseng, has long been recognized for its therapeutic effects on cardiovascular diseases. Saponins, including ginsenosides and notoginsenosides, are the main bioactive components of P. notoginseng. The biosynthesis of saponins is closely related to the defense responses orchestrated by endogenous hormones., Results: To provide new insights into the underlying role of phytohormone jasmonic acid (JA) in the synthesis and regulation of saponins, we performed an ultra-performance liquid chromatography analysis of different tissues of P. notoginseng aged 2-4 years. Moreover, by combined evaluation of saponin content and transcriptome profiling of each tissue, the spatial and temporal distribution of saponins was analyzed. N notoginsenoside R1, ginsenoside Rb1 and ginsenoside Rd accumulated in the underground tissues, including the root, tuqi, fibril and rhizome. In agreement with this data, the corresponding genes of the endogenous hormone JAs, especially coronatine insensitive 1 (COI1) and myelocytomatosis proteins 2 (MYC2), were predominantly expressed in the underground tissues. The tissue- and age-specific distribution of saponins was consistent with the expression of genes involved in JA biosynthetic, metabolic and signaling pathways., Conclusion: The present study has revealed the temporal and spatial effects of endogenous phtohormones in the synthesis and regulation of notoginsenosides, which will provide a significant impact on improving the ecological planting technology, cultivating new high-quality varieties and protecting the rare resources of medicinal P. notoginseng. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Acorus tatarinowii Schott is a well-known Chinese traditional herb. Lignin is the major biologically active ingredient and exerts a broad range of pharmacological effects: it is an antitumor, antioxidant and bacteriostatic agent, and protects the cardiovascular system. In the present study, the transcriptomes of the leaf and rhizome tissues of A. tatarinowii Schott were obtained using the BGISEQ-500 platform. A total of 141777 unigenes were successfully assembled, of which 76714 were annotated in public databases. Further analysis of the lignin biosynthesis pathway revealed a total of 107 unigenes encoding 8 key enzymes, which were involved in this pathway. Furthermore, the expression of the key genes involved in lignin synthesis in different tissues was identified by quantitative real-time PCR. Analysis of the differentially expressed genes (DEGs) showed that most of the up-regulated unigenes were enriched in rhizome tissues. In addition, 2426 unigenes were annotated to the transcriptome factor (TF) family. Moreover, 16 TFs regulating the same key enzyme (peroxidase) were involved in the lignin synthesis pathway. The alignment of peroxidase amino acid sequences and the analysis of the structural characteristics revealed that the key peroxidase enzyme had well-conserved sequences, spatial structures, and active sites. The present study is the first to provide comprehensive genetic information on A. tatarinowii Schott at the transcriptional level, and will facilitate our understanding of the lignin biosynthesis pathway., (© 2021 The Author(s).)

The legume-rhizobial symbiosis results in the formation of root nodules that provide an ecological niche for nitrogen-fixing bacteria. However, plant-bacteria genotypic interactions can lead to wide variation in nitrogen fixation efficiency, and it is not uncommon that a bacterial strain forms functional (Fix + ) nodules on one plant genotype but nonfunctional (Fix - ) nodules on another. Host genetic control of this specificity is unknown. We herein report the cloning of the Medicago truncatula NFS1 gene that regulates the fixation-level incompatibility with the microsymbiont Sinorhizobium meliloti Rm41. We show that NFS1 encodes a nodule-specific cysteine-rich (NCR) peptide. In contrast to the known role of NCR peptides as effectors of endosymbionts' differentiation to nitrogen-fixing bacteroids, we demonstrate that specific NCRs control discrimination against incompatible microsymbionts. NFS1 provokes bacterial cell death and early nodule senescence in an allele-specific and rhizobial strain-specific manner, and its function is dependent on host genetic background., Competing Interests: The authors declare no conflict of interest.

Plants produce a large variety of alkaloids, which have diverse chemical structures and biological activities. Many of these alkaloids accumulate in vacuoles. Although some membrane proteins on tonoplasts have been identified as alkaloid uptake transporters, few have been characterized to date, and relatively little is known about the mechanisms underlying alkaloid transport and accumulation in plant cells. Berberine is a model alkaloid. Although all genes involved in berberine biosynthesis, as well as the master regulator, have been identified, the gene responsible for the final accumulation of berberine at tonoplasts has not been determined. This study showed that a multidrug and toxic compound extrusion protein 1 (CjMATE1) may act as a berberine transporter in cultured Coptis japonica cells. CjMATE1 was found to localize at tonoplasts in C. japonica cells and, in intact plants, to be expressed preferentially in rhizomes, the site of abundant berberine accumulation. Cellular transport analysis using a yeast expression system showed that CjMATE1 could transport berberine. Expression analysis showed that RNAi suppression of CjbHLH1, a master transcription factor of the berberine biosynthetic pathway, markedly reduced the expression of CjMATE1 in a manner similar to the suppression of berberine biosynthetic genes. These results strongly suggest that CjMATE1 is the transporter that mediates berberine accumulation in vacuoles., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Adventitious roots (ARs) directly affect lotus seedling growth and product quality because principal root is not well developed. However, the details of AR formation at the molecular level have not been determined in lotus. Therefore, three stages were chosen to identify the change of proteins abundant during rhizome formation, using isobaric tags for relative and absolute quantization coupled with liquid chromatography-tandem mass spectrometry to gain insight into the molecular mechanisms involved in AR formation. We totally obtained 323,375 spectra during AR formation. After filtering to eliminate low-scoring spectra, 66,943 spectra, including 53,106 unique spectra, were identified. These unique spectra matched 28,905 peptides, including 24,992 unique peptides, which were assembled into 6686 proteins. In the C0/C1 and C1/C2 stages, 66 and 32 proteins showed enhanced abundance, and 173 and 73 proteins showed decreased abundance, respectively. Seventeen important AR formation-related proteins from the three stages were identified, and the expressions of nine genes from the above-identified proteins were assessed by qRT-PCR. This article provides a comprehensive understanding of the changes in metabolism during AR formation, and is helpful to accelerate the progress of breeding in fulture in lotus root.

Background: For generations, the rhizomes of Drynaria ferns have been used as traditional medicine in Asia. Despite this, the bioactivities of Drynaria rhizomes and leaves have rarely been studied scientifically., Methods: This study evaluates the antioxidant properties of the methanolic extracts of the fertile fronds and rhizomes from three species in this genus: Drynaria quercifolia, Drynaria rigidula and Drynaria sparsisora. The phenolic and flavonoid contents of the samples were respectively quantified with the total phenolic content (TPC) and total flavonoid content (TFC) assays, while the antioxidant activities were determined via measuring the DPPH radical scavenging activity (FRS), ferric reducing power (FRP), ferrous ion chelating (FIC) activity and lipid peroxidation inhibition (LPI). The tyrosinase inhibition activity of all three species was also reported., Results: The fertile fronds of D. quercifolia were found to exhibit the highest overall TPC (2939 ± 469 mg GAE/100 g) and antioxidant activity amongst all the samples, and the fertile fronds of D. quercifolia and D. rigidula exhibited superior TPC and FRP compared to their rhizomes, despite only the latter being widely used in traditional medicine. The fronds of D. quercifolia had high tyrosinase inhibition activity (56.6 ± 5.0 %), but most of the Drynaria extracts showed unexpected tyrosinase enhancement instead, particularly for D. sparsisora's fronds., Conclusion: The high bioactivity of the fertile fronds in the fern species indicate that there is value in further research on the fronds of ferns which are commonly used mostly, or only, for their rhizomes.

A novel protein, designated as DOI, isolated from the Chinese yam (Dioscorea opposita Thunb.) could be the first protein drug for the treatment of menopausal syndrome and an alternative to hormone replacement therapy (HRT), which is known to have undesirable side effects. DOI is an acid- and thermo-stable protein with a distinctive N-terminal sequence Gly-Ile-Gly-Lys-Ile-Thr-Thr-Tyr-Trp-Gly-Gln-Tyr-Ser-Asp-Glu-Pro-Ser-Leu-Thr-Glu. DOI was found to stimulate estradiol biosynthesis in rat ovarian granulosa cells; induce estradiol and progesterone secretion in 16- to 18-month-old female Sprague Dawley rats by upregulating expressions of follicle-stimulating hormone receptor and ovarian aromatase; counteract the progression of osteoporosis and augment bone mineral density; and improve cognitive functioning by upregulating protein expressions of brain-derived neurotrophic factor and TrkB receptors in the prefrontal cortex. Furthermore, DOI did not stimulate the proliferation of breast cancer and ovarian cancer cells, which suggest it could be a more efficacious and safer alternative to HRT., Competing Interests: Findings from this study have been used to apply for patents (Patent Cooperation Treaty Patent Pub. No.: WO/2013/023617; U.S. Patent Pub. No.: US20130217626 A1; European Patent Pub. No.: EP 2750684 A1; China Patent pub. No.: CN103945854 A)

The rhizome is responsible for the invasiveness and competitiveness of many plants with great economic and agricultural impact worldwide. Besides its value as an invasive organ, the rhizome plays a role in the establishment and massive growth of forage, providing biomass for biofuel production. Despite these features, little is known about the molecular mechanisms that contribute to rhizome growth, development, and function in plants. In this work, we characterized the proteome of rhizome apical tips and elongation zones from different species using a GeLC-MS/MS (one-dimensional electrophoresis in combination with liquid chromatography coupled online with tandem mass spectrometry) spectral-counting proteomics strategy. Five rhizomatous grasses and an ancient species were compared to study the protein regulation in rhizomes. An average of 2200 rhizome proteins per species were confidently identified and quantified. Rhizome-characteristic proteins showed similar functional distributions across all species analyzed. The over-representation of proteins associated with central roles in cellular, metabolic, and developmental processes indicated accelerated metabolism in growing rhizomes. Moreover, 61 rhizome-characteristic proteins appeared to be regulated similarly among analyzed plants. In addition, 36 showed conserved regulation between rhizome apical tips and elongation zones across species. These proteins were preferentially expressed in rhizome tissues regardless of the species analyzed, making them interesting candidates for more detailed investigative studies about their roles in rhizome development.

Faba bean/maize intercropping significantly promotes maize productivity in phosphorus-deficient soils. This has been attributed to the below-ground interactions including rhizosphere effects and spatial effects. Nevertheless, the molecular mechanisms underlying these interactions have been scarcely investigated. Here, three types of pots were used to distinguish the influences of rhizosphere effects vs. spatial effects. Phosphorus and nitrogen uptake of shoots, biomass, total root length, and root classification were evaluated between the three treatments. Quantitative RT-PCR and proteomics analyses were conducted to investigate the putative components in the molecular basis of these interactions. Quantitative RT-PCR results indicated that rhizosphere effects promoted maize phosphorus status at molecular levels. 66 differentially accumulated protein spots were successfully identified through proteomics analyses. Most of the protein species were found to be involved in phosphorus, nitrogen, and allelochemical metabolism, signal transduction, or stress resistance. The results suggest that rhizosphere effects promoted phosphorus and nitrogen assimilation in maize roots and thus enhanced maize growth and nutrient uptake. The reprogramming of proteome profiles suggests that rhizosphere effects can also enhance maize tolerance through regulating the metabolism of allelochemicals and eliciting systemic acquired resistance via the stimulation of a mitogen-activated protein kinase signal pathway., Biological Significance: The results obtained contribute to a comprehensive understanding of the response of maize to the changes of rhizosphere condition influenced by the below-ground interactions in faba bean/maize intercropping at molecular levels. The identified protein species involved in nutrient metabolisms and stress resistance reveal the molecular basis underlying the major advantages of effective nutrient utilization and higher stress tolerance in legume/cereal intercropping systems. This work provides essential new insights into the putative components in the molecular basis of interspecific facilitation for maize in faba bean/maize intercropping., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Intercropping is an important and sustainable cropping practice in agroecosystems. Peanut/maize intercropping is known to improve the iron (Fe) content of peanuts in calcareous soils. In this study, a proteomic approach was used to uncover the ecological significance of peanut/maize intercropping at the molecular level. We demonstrate that photosynthesis-related proteins accumulated in intercropped peanut leaves, suggesting that the intercropped peanuts had a stronger photosynthetic efficiency. Moreover, stress-response proteins displayed elevated expression levels in both peanut and maize in a monocropping system. This indicated that intercropping contributes to resistance to stress conditions. Allene oxide synthase and 12-oxo-phytodienoic acid reductase, two key enzymes in jasmonate (JA) biosynthesis, increased in abundance in the maize roots of the intercropping system, consistent with the upregulation of JA-induced proteins shown by microarray analysis. These results imply that JA may act as a signaling molecule, playing an important role in intercropping through rhizosphere interaction. This study suggests that peanut/maize intercropping results in high Fe availability in the rhizosphere, leading to variation in the proteins related to carbon and nitrogen metabolism. The advantages of intercropping systems may improve the ecological adaptation of plants to environmental stress., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Bamboo (Phyllostachys praecox) is one of the largest members of the grass family Poaceae, and is one of the most economically important crops in Asia. However, complete knowledge of bamboo development and its molecular mechanisms is still lacking. In the present study, the differences in anatomical structure among rhizome buds, rhizome shoots, and bamboo shoots were compared, and several genes related to the development of the bamboo rhizome bud were identified. The rice cross-species microarray hybridization showed a total of 318 up-regulated and 339 down-regulated genes, including those involved in regulation and signalling, metabolism, and stress, and also cell wall-related genes, in the bamboo rhizome buds versus the leaves. By referring to the functional dissection of the homologous genes from Arabidopsis and rice, the putative functions of the 52 up-regulated genes in the bamboo rhizome bud were described. Six genes related to the development of the bamboo rhizome bud were further cloned and sequenced. These show 66-90% nucleotide identity and 68-98% amino acid identity with the homologous rice genes. The expression patterns of these genes revealed significant differences in rhizome shoots, rhizome buds, bamboo shoots, leaves, and young florets. Furthermore, in situ hybridization showed that the PpRLK1 gene is expressed in the procambium and is closely related to meristem development of bamboo shoots. The PpHB1 gene is expressed at the tips of bamboo shoots and procambium, and is closely related to rhizome bud formation and procambial development. To our knowledge, this is the first report that uses rice cross-species hybridization to identify genes related to bamboo rhizome bud development, and thereby contributes to the further understanding of the molecular mechanism involved in bamboo rhizome bud development.

The interface between the host cell and the microsymbiont is an important zone for development and differentiation during successive stages of the rhizobium-legume symbiosis. Legume root nodule extensins, otherwise known as arabinogalactanprotein-extensin (AGPE) are abundant components of the infection thread matrix. We have characterized the origin and distribution of these glycoproteins at the symbiotic interface of root nodules of symbiotically defective mutants of pea (Pisum sativum L.) using immunogold localization with MAC265 an anti-AGPE monoclonal antibody. For mutants with defective growth of infection threads, the AGPE epitope was abundant in the extracellular matrix surrounding infected host cells in the central infected tissue of the nodule as well as being present in the lumen of Rhizobium-induced infection threads. This suggests a mis-targetting of AGPE as a consequence of abnormal growth of the infection threads. Furthermore, mutants in gene sym33 showed reduced labelling with MAC265, and in infection threads and droplets label was completely absent, a phenomenon not observed in wild-type nodules. This suggests an alteration in the composition of the infection thread matrix for sym33 mutants which may be correlated with the absence of endocytosis of rhizobia into the host cytoplasm.

The establishment of the symbiosis between legume plants and rhizobial bacteria depends on the production of rhizobial lipo-chitooligosaccharidic signals (the Nod factors) that are specifically recognized by roots of the host plant. In Medicago truncatula, specific recognition of Sinorhizobium meliloti and its Nod factors requires the NFP (Nod factor perception) gene, which encodes a putative serine/threonine receptor-like kinase (RLK). The extracellular region of this protein contains three tandem lysin motifs (LysMs), a short peptide domain that is implicated in peptidoglycan or chitin binding in various bacterial or eukaryotic proteins, respectively. We report here the homology modeling of the three LysM domains of M. truncatula NFP based on the structure of a LysM domain of the Escherichia coli membrane-bound lytic murein transglycosidase D (MltD). Expression of NFP in a homologous system (M. truncatula roots) revealed that the protein is highly N-glycosylated, probably with both high-mannose and complex glycans. Surface analysis and docking calculations performed on the models of the three domains were used to predict the most favored binding modes for chitooligosaccharides and Nod factors. A convergent model can be proposed where the sulfated, O-acetylated lipo-chitooligosaccharidic Nod factor of S. meliloti binds in similar orientation to the three LysM domains of M. truncatula NFP. N-glycosylation is not expected to interfere with Nod factor binding in this orientation.