NnSUS1 encodes a sucrose synthase involved in sugar accumulation in lotus seed cotyledons.

Fresh lotus seeds are gaining favor with consumers for their crunchy texture and natural sweetness. However, the intricacies of sugar accumulation in lotus seeds remain elusive, which greatly hinders the quality improvement of fresh lotus seeds. This study endeavors to elucidate this mechanism by identifying and characterizing the sucrose synthase (SUS) gene family in lotus. Comprising five distinct members, namely NnSUS1 to NnSUS5, each gene within this family features a C-terminal glycosyl transferase1 (GT1) domain. Among them, NnSUS1 is the predominately expressed gene, showing high transcript abundance in the floral organs and cotyledons. NnSUS1 was continuously up-regulated from 6 to 18 days after pollination (DAP) in lotus cotyledons. Furthermore, NnSUS1 demonstrates co-expression relationships with numerous genes involved in starch and sucrose metabolism. To investigate the function of NnSUS1, a transient overexpression system was established in lotus cotyledons, which confirmed the gene's contribution to sugar accumulation. Specifically, transient overexpression of NnSUS1 in seed cotyledons leads to a significant increase in the levels of total soluble sugar, including sucrose and fructose. These findings provide valuable theoretical insights for improving sugar content in lotus seeds through molecular breeding methods.

Nitrogen and Nod factor signaling determine Lotus japonicus root exudate composition and bacterial assembly.

Symbiosis with soil-dwelling bacteria that fix atmospheric nitrogen allows legume plants to grow in nitrogen-depleted soil. Symbiosis impacts the assembly of root microbiota, but it is unknown how the interaction between the legume host and rhizobia impacts the remaining microbiota and whether it depends on nitrogen nutrition. Here, we use plant and bacterial mutants to address the role of Nod factor signaling on Lotus japonicus root microbiota assembly. We find that Nod factors are produced by symbionts to activate Nod factor signaling in the host and that this modulates the root exudate profile and the assembly of a symbiotic root microbiota. Lotus plants with different symbiotic abilities, grown in unfertilized or nitrate-supplemented soils, display three nitrogen-dependent nutritional states: starved, symbiotic, or inorganic. We find that root and rhizosphere microbiomes associated with these states differ in composition and connectivity, demonstrating that symbiosis and inorganic nitrogen impact the legume root microbiota differently. Finally, we demonstrate that selected bacterial genera characterizing state-dependent microbiomes have a high level of accurate prediction.

RinRK1 enhances NF receptors accumulation in nanodomain-like structures at root-hair tip.

Legume-rhizobia root-nodule symbioses involve the recognition of rhizobial Nod factor (NF) signals by NF receptors, triggering both nodule organogenesis and rhizobial infection. RinRK1 is induced by NF signaling and is essential for infection thread (IT) formation in Lotus japonicus. However, the precise mechanism underlying this process remains unknown. Here, we show that RinRK1 interacts with the extracellular domains of NF receptors (NFR1 and NFR5) to promote their accumulation at root hair tips in response to rhizobia or NFs. Furthermore, Flotillin 1 (Flot1), a nanodomain-organizing protein, associates with the kinase domains of NFR1, NFR5 and RinRK1. RinRK1 promotes the interactions between Flot1 and NF receptors and both RinRK1 and Flot1 are necessary for the accumulation of NF receptors at root hair tips upon NF stimulation. Our study shows that RinRK1 and Flot1 play a crucial role in NF receptor complex assembly within localized plasma membrane signaling centers to promote symbiotic infection.

Upregulated synthesis and production of bioactive compounds in Lotus arabicus L. by in vitro feeding with dried powder of date palm seeds.

BACKGROUND: Plants are considered the primary source of many principal bioactive compounds that have been utilized in a wide range of applications including the pharmaceutical and biotechnological industries. Therefore, there is an imperative need to modulate the production of natural bioactive components. The present study aimed to determine the importance of dried and pulverized date palm seeds (DPS) as a natural elicitor for the synthesis of secondary metabolites in Lotus arabicus L. RESULTS: The presence of various antioxidant compounds, simple sugars, amino acids, fatty acids and reasonable mineral contents was distinct in the phytochemical characterization of DPS. The major components detected in DPS analysis were the 5-(hydroxymethyl) furfural and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone. The induced callus of L. arabicus (seven weeks old) was supplemented with DPS at different concentrations (0, 2, 4, 8 and 10 g/l) in culture media. Treatment with 8 g/l DPS induced the highest antioxidant capacity, ascorbic acid content and secondary metabolites (total phenolics and flavonoids) in the produced callus. Stress biomarkers (hydrogen peroxide and malondialdehyde) were found in the control ranges except at 10 g/l DPS. The expression patterns of key genes involoved in secondary metabolism modulation, such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS) and deoxyxylulose phosphate reductoisomerase (DXR), were triggered after DPS treatments. Moreover, the quantitative profiling of phenolic and flavonoid compounds showed that supplementation with DPS, especially at 8 g/l, led to pronounced increases in most of the measured compounds. CONCLUSION: The marked upregulation of eliciting-responsive genes and overproduction of secondary metabolites provide molecular-based evidence for intensifying the principal pathways of phenylpropanoid, flavonoid and terpenoid biosynthesis. Overall, the present in vitro study highlights the stimulating capacity of DPS utilization to improve the bioactive components of L. arabicus at the physiological and molecular levels, enhancing its potential as a medicinal herb.

NnARF17 and NnARF18 from lotus promote root formation and modulate stress tolerance in transgenic Arabidopsis thaliana.

Auxin response factors (ARFs) play a crucial role in regulating gene expression within the auxin signal transduction pathway, particularly during adventitious root (AR) formation. In this investigation, we identified full-length sequences for ARF17 and ARF18, encompassing 1,800 and 2,055 bp, encoding 599 and 684 amino acid residues, respectively. Despite exhibiting low sequence homology, the ARF17- and ARF18-encoded proteins displayed significant structural similarity and shared identical motifs. Phylogenetic analysis revealed close relationships between NnARF17 and VvARF17, as well as NnARF18 and BvARF18. Both ARF17 and ARF18 demonstrated responsiveness to exogenous indole-3-acetic acid (IAA), ethephon, and sucrose, exhibiting organ-specific expression patterns. Beyond their role in promoting root development, these ARFs enhanced stem growth and conferred drought tolerance while mitigating waterlogging stress in transgenic Arabidopsis plants. RNA sequencing data indicated upregulation of 51 and 75 genes in ARF17 and ARF18 transgenic plants, respectively, including five and three genes associated with hormone metabolism and responses. Further analysis of transgenic plants revealed a significant decrease in IAA content, accompanied by a marked increase in abscisic acid content under normal growth conditions. Additionally, lotus seedlings treated with IAA exhibited elevated levels of polyphenol oxidase, IAA oxidase, and peroxidase. The consistent modulation of IAA content in both lotus and transgenic plants highlights the pivotal role of IAA in AR formation in lotus seedlings.

Effect of lotus seed resistant starch on the bioconversion pathway of taurocholic acid by regulating the intestinal microbiota.

Taurocholic acid (TCA) is abundant in the rat intestine and has multiple health benefits. In the gut, intestinal microbiota can transform TCA into different bile acid (BA) derivatives, with the composition of microbiota playing a crucial role in the transformation process. This study aims to investigate how lotus seed resistant starch (LRS) can regulate microbiota to influence BA transformation. A fecal fermentation study was conducted in vitro, using either LRS, high-amylose maize starch (HAMS), or glucose (GLU) to analyze microbiota composition, BA content, and metabolic enzyme activities over different fermentation times. Bioinformatics analysis found that LRS increased the relative abundance of Enterococcus, Bacillus, and Lactobacillus, and decreased Escherichia-Shigella, compared with HAMS and GLU. LRS also reduced total BA content and accelerated the conversion of TCA to cholic acid, deoxycholic acid, and other derivatives. These results reveal that LRS and GLU tend to mediate the dehydroxy pathway, whereas HAMS tends to secrete metabolic enzymes in the epimerization pathway. Therefore, the evidence that LRS may regulate TCA bioconversion may benefit human colon health research and provide an important theoretical basis, as well as offer new concepts for the development of functional foods.

Aquaporin LjNIP1;5 positively modulates drought tolerance by promoting arbuscular mycorrhizal symbiosis in Lotus japonicus.

Drought stress often affects crop growth and even causes crop death, while aquaporins can maintain osmotic balance by transporting water across membranes, so it is important to study how to improve drought tolerance of crops by using aquaporins. In this work, we characterize a set of subfamily members named NIPs belonging to the family of aquaporins in Lotus japonicus, grouping 14 family members based on the sequence similarity in the aromatic/arginine (Ar/R) region. Among these members, LjNIP1;5 is one of the genes with the highest expression in roots which is induced by the AM fungus. In Lotus japonicus, LjNIP1;5 is highly expressed in symbiotic roots, and its promoter can be induced by drought stress and AM fungus. Root colonization analysis reveals that ljnip1:5 mutant exhibits lower mycorrhizal colonization than the wild type, with increasing the proportion of large arbuscule, and fewer arbuscule produced by symbiosis under drought stress. In the LjNIP1;5OE plant, we detected a strong antioxidant capacity compared to the control, and LjNIP1;5OE showed higher stem length under drought stress. Taken together, the current results facilitate our comprehensive understanding of the plant adaptive to drought stress with the coordination of the specific fungi.

Biochemical and Structural Studies of LjSK1, a Lotus japonicus GSK3ß/SHAGGY-like Kinase, Reveal Its Functional Role.

The crystal structure of a truncated form of the Lotus japonicus glycogen synthase kinase 3ß (GSK3ß) like kinase (LjSK190-467) has been resolved at 2.9 Å resolution, providing, for the first time, structural data for a plant GKS3ß like kinase. The 3D structure of LjSK190-467 revealed conservation at the structural level for this plant member of the GSK3ß family. However, comparative structural analysis to the human homologue revealed significant differences at the N- and C-termini, supporting the notion for an additional regulatory mechanism in plant GSK3-like kinases. Structural similarities at the catalytic site and the ATP binding site explained the similarity in the function of the human and plant protein. LjSK1 and lupeol are strongly linked to symbiotic bacterial infection and nodulation initiation. An inhibitory capacity of lupeol (IC50 = 0.77 µM) for LjSK1 was discovered, providing a biochemical explanation for the involvement of these two molecules in nodule formation, and constituted LjSK1 as a molecular target for the discovery of small molecule modulators for crop protection and development. Studies on the inhibitory capacity of two phytogenic triterpenoids (betulinic acid and hederacoside C) to LjSK1 provided their structure-activity relationship and showed that hederacoside C can be the starting point for such endeavors.

Long- and short-read sequencing methods discover distinct circular RNA pools in Lotus japonicus.

Circular RNAs (circRNAs) are covalently closed single-stranded RNAs, generated through a back-splicing process that links a downstream 5' site to an upstream 3' end. The only distinction in the sequence between circRNA and their linear cognate RNA is the back splice junction. Their low abundance and sequence similarity with their linear origin RNA have made the discovery and identification of circRNA challenging. We have identified almost 6000 novel circRNAs from Lotus japonicus leaf tissue using different enrichment, amplification, and sequencing methods as well as alternative bioinformatics pipelines. The different methodologies identified different pools of circRNA with little overlap. We validated circRNA identified by the different methods using reverse transcription polymerase chain reaction and characterized sequence variations using nanopore sequencing. We compared validated circRNA identified in L. japonicus to other plant species and showed conservation of high-confidence circRNA-expressing genes. This is the first identification of L. japonicus circRNA and provides a resource for further characterization of their function in gene regulation. CircRNAs identified in this study originated from genes involved in all biological functions of eukaryotic cells. The comparison of methodologies and technologies to sequence, identify, analyze, and validate circRNA from plant tissues will enable further research to characterize the function and biogenesis of circRNA in L. japonicus.

IMA peptides regulate root nodulation and nitrogen homeostasis by providing iron according to internal nitrogen status.

Legumes control root nodule symbiosis (RNS) in response to environmental nitrogen availability. Despite the recent understanding of the molecular basis of external nitrate-mediated control of RNS, it remains mostly elusive how plants regulate physiological processes depending on internal nitrogen status. In addition, iron (Fe) acts as an essential element that enables symbiotic nitrogen fixation; however, the mechanism of Fe accumulation in nodules is poorly understood. Here, we focus on the transcriptome in response to internal nitrogen status during RNS in Lotus japonicus and identify that IRON MAN (IMA) peptide genes are expressed during symbiotic nitrogen fixation. We show that LjIMA1 and LjIMA2 expressed in the shoot and root play systemic and local roles in concentrating internal Fe to the nodule. Furthermore, IMA peptides have conserved roles in regulating nitrogen homeostasis by adjusting nitrogen-Fe balance in L. japonicus and Arabidopsis thaliana. These findings indicate that IMA-mediated Fe provision plays an essential role in regulating nitrogen-related physiological processes.

Regulation of nitric oxide by phytoglobins in Lotus japonicus is involved in mycorrhizal symbiosis with Rhizophagus irregularis.

Various reactive molecular species are generated in plant-microbe interactions, and these species participate in defense and symbiotic responses. Leguminous plants successfully establish symbiosis by maintaining an appropriate level of nitric oxide (NO), which is generated in the roots and nodules during root nodule symbiosis. Phytoglobin (plant hemoglobin) controls NO levels in plants. In this study, we investigated mycorrhizal symbiosis, which occurs in more than 80% of land plants, between Rhizophagus irregularis and Lotus japonicus to clarify the involvement of phytoglobin-mediated NO regulation. The mycorrhizae of L. japonicus exhibited higher NO levels in the presence of R. irregularis than in its absence, especially at the infection site. LjGlb1-1, a phytoglobin that regulates NO level in L. japonicus, was upregulated during symbiosis with R. irregularis. In transformed hairy roots carrying the ProLjGlb1-1:GUS construct, LjGlb1-1 expression was observed at the R. irregularis infection site. We further examined the symbiotic phenotypes of L. japonicus lines with high and low LjGlb1-1 expression with R. irregularis. During mycorrhizal symbiosis, the high LjGlb1-1 expression line exhibited better growth than the wild-type, whereas the low expression line exhibited poor growth. In addition, the expression of LjPT4, a phosphate transporter specific to mycorrhizal symbiosis, was higher in the high LjGlb1-1 expression line, whereas that of the tubulin gene of R. irregularis was lower in the low LjGlb1-1 expression line than in the wild-type. These results confirm that NO regulation by LjGlb1-1 is involved in mycorrhizal symbiosis in L. japonicus, as it is reportedly in nitrogen-fixing symbiosis.

Spatially and temporally distinct Ca2+ changes in Lotus japonicus roots orient fungal-triggered signalling pathways towards symbiosis or immunity.

Plants activate an immune or symbiotic response depending on the detection of distinct signals from root-interacting microbes. Both signalling cascades involve Ca2+ as a central mediator of early signal transduction. In this study, we combined aequorin- and cameleon-based methods to dissect the changes in cytosolic and nuclear Ca2+ concentration caused by different chitin-derived fungal elicitors in Lotus japonicus roots. Our quantitative analyses highlighted the dual character of the evoked Ca2+ responses taking advantage of the comparison between different genetic backgrounds: an initial Ca2+ influx, dependent on the LysM receptor CERK6 and independent of the common symbiotic signalling pathway (CSSP), is followed by a second CSSP-dependent and CERK6-independent phase, that corresponds to the well-known perinuclear/nuclear Ca2+ spiking. We show that the expression of immunity marker genes correlates with the amplitude of the first Ca2+ change, depends on elicitor concentration, and is controlled by Ca2+ storage in the vacuole. Our findings provide an insight into the Ca2+-mediated signalling mechanisms discriminating plant immunity- and symbiosis-related pathways in the context of their simultaneous activation by single fungal elicitors.

Functional characterization of polyol/monosaccharide transporter 1 in Lotus japonicus.

Polyol/Monosaccharide Transporters (PLTs/PMTs) localized in the plasma membrane have previously been identified in plants. The physiological role and the functional properties of these proteins in legume plants are, however, unclear. Here we describe the functional analysis of LjPLT1, a plasma membrane-localized PLT protein from Lotus japonicus. The LjPLT1 gene was strongly expressed in the vascular tissue of roots, stems and leaves. Expression of the LjPLT1 cDNAs in yeast revealed that the protein functions as a broad-spectrum H+ -symporter for both linear polyols of sorbitol and mannitol, and cyclic polyol myo-inositol. It also catalyzes the transport of different hexoses, including fructose, glucose, galactose and mannose. Overexpression of LjPLT1 (OELjPLT1) results in inhibition of plant growth and a decrease in nodule nitrogenase activity in L. japonicus. The soluble sugars were increased in newly expanded leaves, roots and nodules but decreased in mature leaves in OELjPLT1 plants. In addition, the OELjPLT1 seedlings displayed an increased sensitivity to high content mannitol and boron toxicity, but neither drought nor salinity stresses. Taken together, the present study indicates that the LjPLT1 protein may participate in the translocation of hexoses/polyols to regulate multiple physiological and growth processes in L. japonicus.

Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp.

MAIN CONCLUSION: The complexes involving MYBPA2, TT2b, and TT8 proteins are the critical regulators of ANR and LAR genes to promote the biosynthesis of proanthocyanidins in the leaves of Lotus spp. The environmental impact and health of ruminants fed with forage legumes depend on the herbage's concentration and structure of proanthocyanidins (PAs). Unfortunately, the primary forage legumes (alfalfa and clover) do not contain substantial levels of PAs. No significant progress has been made to induce PAs to agronomically valuable levels in their edible organs by biotechnological approaches thus far. Building this trait requires a profound knowledge of PA regulators and their interplay in species naturally committed to accumulating these metabolites in the target organs. Against this background, we compared the shoot transcriptomes of two inter-fertile Lotus species, namely Lotus tenuis and Lotus corniculatus, polymorphic for this trait, to search for differentially expressed MYB and bHLH genes. We then tested the expression of the above-reported regulators in L. tenuis x L. corniculatus interspecific hybrids, several Lotus spp., and different L. corniculatus organs with contrasting PA levels. We identified a novel MYB activator and MYB-bHLH-based complexes that, when expressed in Nicotiana benthamiana, trans-activated the promoters of L. corniculatus anthocyanidin reductase and leucoanthocyanidin reductase 1 genes. The last are the two critical structural genes for the biosynthesis of PAs in Lotus spp. Competition between MYB activators for the transactivation of these promoters also emerged. Overall, by employing Lotus as a model genus, we refined the transcriptional network underlying PA biosynthesis in the herbage of legumes. These findings are crucial to engineering this trait in pasture legumes.

Mutation of MYB36 affects isoflavonoid metabolism, growth, and stress responses in Lotus japonicus.

Isoflavonoids are mostly produced by legumes although little is known about why and how legumes are able to regulate the biosynthesis of these particular compounds. Understanding the role of potential regulatory genes of the isoflavonoid biosynthetic pathway constitutes an important topic of research. The LORE1 mutation of the gene encoding the transcription factor MYB36 allowed the identification of this gene as a regulator of isoflavonoid biosynthesis in Lotus japonicus plants. The levels of several isoflavonoid compounds were considerably lower in two lines of Ljmyb36 mutant plants compared to the WT. In addition, we found that Ljmyb36 mutant plants were significantly smaller and showed a substantial decrease in the chlorophyll levels under normal growth conditions. The analysis of plants subjected to different types of abiotic stress conditions further revealed that mutant plants presented a higher sensitivity than WT plants, indicating that the MYB36 transcription factor is also involved in the stress response in L. japonicus plants.

NnWOX1-1, NnWOX4-3, and NnWOX5-1 of lotus (Nelumbo nucifera Gaertn)promote root formation and enhance stress tolerance in transgenic Arabidopsis thaliana.

BACKGROUND: Adventitious roots (ARs) represent an important organ system for water and nutrient uptake in lotus plants because of degeneration of the principal root. The WUSCHEL-related homeobox (WOX) gene regulates plant development and growth by affecting the expression of several other genes. In this study, three WOX genes, NnWOX1-1, NnWOX4-3, and NnWOX5-1, were isolated and their functions were assessed in Arabidopsis plants. RESULTS: The full lengths of NnWOX1-1, NnWOX4-3, and NnWOX5-1 were 1038, 645, and 558 bp, encoding 362, 214, and 185 amino acid residues, respectively. Phylogenetic analysis classified NnWOX1-1 and NnWOX4-3 encoding proteins into one group, and NnWOX5-1 and MnWOX5 encoding proteins exhibited strong genetic relationships. The three genes were induced by sucrose and indoleacetic acid (IAA) and exhibited organ-specific expression characteristics. In addition to improving root growth and salt tolerance, NnWOX1-1 and NnWOX4-3 promoted stem development in transgenic Arabidopsis plants. A total of 751, 594, and 541 genes, including 19, 19, and 13 respective genes related to ethylene and IAA metabolism and responses, were enhanced in NnWOX1-1, NnWOX4-3, and NnWOX5-1 transgenic plants, respectively. Further analysis showed that ethylene production rates in transgenic plants increased, whereas IAA, peroxidase, and lignin content did not significantly change. Exogenous application of ethephon on lotus seedlings promoted AR formation and dramatically increased the fresh and dry weights of the plants. CONCLUSIONS: NnWOX1-1, NnWOX4-3, and NnWOX5-1 influence root formation, stem development, and stress adaptation in transgenic Arabidopsis plants by affecting the transcription of multiple genes. Among these, changes in gene expression involving ethylene metabolism and responses likely critically affect the development of Arabidopsis plants. In addition, ethylene may represent an important factor affecting AR formation in lotus seedlings.

Single-cell analysis identifies genes facilitating rhizobium infection in Lotus japonicus.

Legume-rhizobium signaling during establishment of symbiotic nitrogen fixation restricts rhizobium colonization to specific cells. A limited number of root hair cells allow infection threads to form, and only a fraction of the epidermal infection threads progress to cortical layers to establish functional nodules. Here we use single-cell analysis to define the epidermal and cortical cell populations that respond to and facilitate rhizobium infection. We then identify high-confidence nodulation gene candidates based on their specific expression in these populations, pinpointing genes stably associated with infection across genotypes and time points. We show that one of these, which we name SYMRKL1, encodes a protein with an ectodomain predicted to be nearly identical to that of SYMRK and is required for normal infection thread formation. Our work disentangles cellular processes and transcriptional modules that were previously confounded due to lack of cellular resolution, providing a more detailed understanding of symbiotic interactions.

Metabolite profiling and screening of callus browning-related genes in lotus (Nelumbo nucifera).

Callus browning is a major drawback to lotus callus proliferation and regeneration. However, the underlying mechanism of its formation remains largely unknown. Herein, we aimed to explore the metabolic and molecular basis of lotus callus browning by combining histological staining, high-throughput metabolomics, and transcriptomic assays for lotus callus at three browning stages. Histological stained brown callus cross sections displayed severe cell death symptoms, accompanied by an obvious accumulation of polyphenols and lignified materials. Widely targeted metabolomics revealed extensively decreased accumulation of most detected flavonoids and benzylisoquinoline alkaloids (BIAs), as well as a few phenolic acids, amino acids and their derivatives in callus with browning symptoms. Conversely, the contents of most detected tannins were significantly increased. Subsequent comparative transcriptomics identified a set of differentially expressed genes (DEGs) associated with the biosynthesis and regulation of flavonoids and BIAs in lotus. Notably, callus browning was coupled with significantly up-regulated expression of two polyphenol oxidase (PPO) and 17 peroxidase (POD) encoding genes, while the expression of ethylene associated genes remained at marginal levels. These results suggest that lotus callus browning is primarily controlled at the level of metabolism, wherein the oxidation of flavonoids and BIAs is crucially decisive.

Primary and Secondary Metabolites in Lotus japonicus.

Lotus japonicus is a leguminous model plant used to gain insight into plant physiology, stress response, and especially symbiotic plant-microbe interactions, such as root nodule symbiosis or arbuscular mycorrhiza. Responses to changing environmental conditions, stress, microbes, or insect pests are generally accompanied by changes in primary and secondary metabolism to account for physiological needs or to produce defensive or signaling compounds. Here we provide an overview of the primary and secondary metabolites identified in L. japonicus to date. Identification of the metabolites is mainly based on mass spectral tags (MSTs) obtained by gas chromatography linked with tandem mass spectrometry (GC-MS/MS) or liquid chromatography-MS/MS (LC-MS/MS). These MSTs contain retention index and mass spectral information, which are compared to databases with MSTs of authentic standards. More than 600 metabolites are grouped into compound classes such as polyphenols, carbohydrates, organic acids and phosphates, lipids, amino acids, nitrogenous compounds, phytohormones, and additional defense compounds. Their physiological effects are briefly discussed, and the detection methods are explained. This review of the exisiting literature on L. japonicus metabolites provides a valuable basis for future metabolomics studies.

ChIFNα regulates adventitious root development in Lotus japonicus via an auxin-mediated pathway.

Adventitious roots (ARs), developing from non-root tissue, play an important role in some plants. Here, the molecular mechanism of AR differentiation in Lotus japonicus L. (L. japonicus) with the transformed chicken interferon alpha gene (ChIFNα) encoding cytokine was studied. ChIFNα transgenic plants (TP) were identified by GUS staining, PCR, RT-PCR, and ELISA. Up to 0.175 µg/kg rChIFNα was detected in TP2 lines. Expressing rChIFNα promotes AR development by producing longer roots than controls. We found that the effect was enhanced with the auxin precursor IBA treatment in TP. IAA contents, POD, and PPO activities associated with auxin regulation were higher than wild type (WT) in TP and exogenous ChIFNα treatment plants. Transcriptome analysis revealed 48 auxin-related differentially expressed genes (DEGs) (FDR < 0.05), which expression levels were verified by RT-qPCR analysis. GO enrichment analysis of DEGs also highlighted the auxin pathway. Further analysis found that ChIFNα significantly enhanced auxin synthesis and signaling mainly with up-regulated genes of ALDH, and GH3. Our study reveals that ChIFNα can promote plant AR development by mediating auxin regulation. The findings help explore the role of ChIFNα cytokines and expand animal gene sources for the molecular breeding of growth regulation of forage plants.