Diverse branching forms regulated by a core auxin transport mechanism in plants.

Diverse branching forms have evolved multiple times across the tree of life to facilitate resource acquisition and exchange with the environment. In the vascular plant group, the ancestral pattern of branching involves dichotomy of a parent shoot apex to form two new daughter apices. The molecular basis of axillary branching in Arabidopsis is well understood, but few regulators of dichotomous branching are known. Through analyses of dichotomous branching in the lycophyte, Selaginella kraussiana, we identify PIN-mediated auxin transport as an ancestral branch regulator of vascular plants. We show that short-range auxin transport out of the apices promotes dichotomy and that branch dominance is globally coordinated by long-range auxin transport. Uniquely in Selaginella, angle meristems initiate at each dichotomy, and these can develop into rhizophores or branching angle shoots. We show that long-range auxin transport and a transitory drop in PIN expression are involved in angle shoot development. We conclude that PIN-mediated auxin transport is an ancestral mechanism for vascular plant branching that was independently recruited into Selaginella angle shoot development and seed plant axillary branching during evolution.

Structure-function analysis of plant G-protein regulatory mechanisms identifies key Gα-RGS protein interactions.

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.

The Possible Earliest Allopolyploidization in Tracheophytes Revealed by Phylotranscriptomics and Morphology of Selaginellaceae.

Selaginellaceae, originated in the Carboniferous and survived the Permian-Triassic mass extinction, is the largest family of lycophyte, which is sister to other tracheophytes. It stands out from tracheophytes by exhibiting extraordinary habitat diversity and lacking polyploidization. The organelle genome-based phylogenies confirmed the monophyly of Selaginella, with six or seven subgenera grouped into two superclades, but the phylogenetic positions of the enigmatic Selaginella sanguinolenta clade remained problematic. Here, we conducted a phylogenomic study on Selaginellaceae utilizing large-scale nuclear gene data from RNA-seq to elucidate the phylogeny and explore the causes of the phylogenetic incongruence of the S. sanguinolenta clade. Our phylogenetic analyses resolved three different positions of the S. sanguinolenta clade, which were supported by the sorted three nuclear gene sets, respectively. The results from the gene flow test, species network inference, and plastome-based phylogeny congruently suggested a probable hybrid origin of the S. sanguinolenta clade involving each common ancestor of the two superclades in Selaginellaceae. The hybrid hypothesis is corroborated by the evidence from rhizophore morphology and spore micromorphology. The chromosome observation and Ks distributions further suggested hybridization accompanied by polyploidization. Divergence time estimation based on independent datasets from nuclear gene sets and plastid genome data congruently inferred that allopolyploidization occurred in the Early Triassic. To our best knowledge, the allopolyploidization in the Mesozoic reported here represents the earliest record of tracheophytes. Our study revealed a unique triad of phylogenetic positions for a hybrid-originated group with comprehensive evidence and proposed a hypothesis for retaining both parental alleles through gene conversion.

Heterologous expression of a lycophyte protein enhances angiosperm seedling vigor.

Seedling vigor is a key agronomic trait that determines juvenile plant performance. Angiosperm seeds develop inside fruits and are connected to the mother plant through vascular tissues. Their formation requires plant-specific genes, such as BREVIS RADIX (BRX) in Arabidopsis thaliana roots. BRX family proteins are found throughout the euphyllophytes but also occur in non-vascular bryophytes and non-seed lycophytes. They consist of four conserved domains, including the tandem BRX domains. We found that bryophyte or lycophyte BRX homologs can only partially substitute for Arabidopsis BRX (AtBRX) because they miss key features in the linker between the BRX domains. Intriguingly, however, expression of a BRX homolog from the lycophyte Selaginella moellendorffii (SmBRX) in an A. thaliana wild-type background confers robustly enhanced root growth vigor that persists throughout the life cycle. This effect can be traced to a substantial increase in seed and embryo size, is associated with enhanced vascular tissue proliferation, and can be reproduced with a modified, SmBRX-like variant of AtBRX. Our results thus suggest that BRX variants can boost seedling vigor and shed light on the activity of ancient, non-angiosperm BRX family proteins.

ACL5 acquired strict thermospermine synthesis activity during the emergence of vascular plants.

Norspermine (Nspm), one of the uncommon polyamines (PAs), was detected in bryophytes and lycophytes; therefore, the aminopropyltransferases involved in the synthesis of Nspm were investigated. The enzymatic activity was evaluated by the transient high expression of various aminopropyltransferase genes in Nicotiana benthamiana, followed by quantification of PA distribution in the leaves using gas chromatography-mass spectrometry. The bryophyte orthologues of ACL5, which is known to synthesise thermospermine (Tspm) in flowering plants, were found to have strong Nspm synthesis activity. In addition, two ACL5 orthologous with different substrate specificities were conserved in Selaginella moellendorffii, one of which was involved in Tspm synthesis and the other in Nspm synthesis. Therefore, further detailed analysis using these two factors revealed that the ß-hairpin structural region consisting of ß-strands 1 and 2 at the N-terminus of ACL5 is involved in substrate specificity. Through functional analysis of a total of 40 ACL5 genes in 33 organisms, including algae, it was shown that ACL5 has changed its substrate specificity several times during plant evolution and diversification. Furthermore, it was strongly suggested that ACL5 acquired strict Tspm synthesis activity during the emergence of vascular plants, especially through major changes around the ß-hairpin structural region.

The Negative Impact of Prolonged Desiccation on the Recovery of Selaginella bryopteris: Insights Into Autophagy and Cellular Protection Strategies.

Desiccation tolerance is a complex biological phenomenon that allows certain plants to survive extreme dehydration and revive upon rehydration. Although significant progress has been made in understanding the physiological and molecular mechanisms involved in desiccation tolerance, recovery mechanisms after prolonged desiccation periods are enigmatic. Combining physiological, biochemical, transcriptomic and metabolomic approaches, we investigated the role of prolonged desiccation on recovery of Selaginella bryopteris. Prolonged desiccation causes a decline in the antioxidant system, leading to accumulation of ROS that hinder recovery by inducing cellular damage. Transcriptome and WGCNA analysis revealed the significance of protective proteins, alternative respiration and protein homeostasis in cellular protection and recovery after short and long-term desiccation. Metabolomic analysis exhibited an increased accumulation of antioxidant compounds, which can be substituted for antioxidant enzymes to maintain cellular protection during prolonged desiccation. The significant role of autophagy and autophagic components was evaluated by H2O2 treatment and phylogenetic analysis of ATG4 and ATG8, which unveiled their substantial role in desiccation tolerance and remarkable conservation of the autophagy-related genes across plant species. Our data demonstrated that prolonged desiccation leads to ROS-induced cell death by extensive autophagy due to enormous loss of protective proteins, antioxidant enzymes and energy resources during desiccation.

The evolution of extremely diverged plastomes in Selaginellaceae (lycophyte) is driven by repeat patterns and the underlying DNA maintenance machinery.

Two factors are proposed to account for the unusual features of organellar genomes: the disruptions of organelle-targeted DNA replication, repair, and recombination (DNA-RRR) systems in the nuclear genome and repetitive elements in organellar genomes. Little is known about how these factors affect organellar genome evolution. The deep-branching vascular plant family Selaginellaceae is known to have a deficient DNA-RRR system and convergently evolved organellar genomes. However, we found that the plastid genome (plastome) of Selaginella sinensis has extremely accelerated substitution rates, a low GC content, pervasive repeat elements, a dynamic network structure, and it lacks direct or inverted repeats. Unexpectedly, its organelle DNA-RRR system is short of a plastid-targeted Recombinase A1 (RecA1) and a mitochondrion-targeted RecA3, in line with other explored Selaginella species. The plastome contains a large collection of short- and medium-sized repeats. Given the absence of RecA1 surveillance, we propose that these repeats trigger illegitimate recombination, accelerated mutation rates, and structural instability. The correlations between repeat quantity and architectural complexity in the Selaginella plastomes support these conclusions. We, therefore, hypothesize that the interplay of the deficient DNA-RRR system and the high repeat content has led to the extraordinary divergence of the S. sinensis plastome. Our study not only sheds new light on the mechanism of plastome divergence by emphasizing the power of cytonuclear integration, but it also reconciles the longstanding contradiction on the effects of DNA-RRR system disruption on genome structure evolution.

Genome-Wide Mining of Selaginella moellendorffii for Hevein-like Lectins and Their Potential Molecular Mimicry with SARS-CoV-2 Spike Glycoprotein.

Multidisciplinary research efforts on potential COVID-19 vaccine and therapeutic candidates have increased since the pandemic outbreak of SARS-CoV-2 in 2019. This search has become imperative due to the increasing emergences and limited widely available medicines. The presence of bioactive anti-SARS-CoV-2 molecules was examined from various plant sources. Among them is a group of proteins called lectins that can bind carbohydrate moieties. In this article, we present ten novel, chitin-specific Hevein-like lectins that were derived from Selaginella moellendorffii v1.0's genome. The capacity of these lectin homologs to bind with the spike protein of SARS-CoV-2 was examined. Using the HDOCK server, 3D-modeled Hevein-domains were docked to the spike protein's receptor binding domain (RBD). The Smo446851, Smo125663, and Smo99732 interacted with Asn343-located complex N-glycan and RBD residues, respectively, with binding free energies of -17.5, -13.0, and -26.5 Kcal/mol. The molecular dynamics simulation using Desmond and the normal-state analyses via torsional coordinate association for the Smo99732-RBD complex using iMODS is characterized by overall higher stability and minimum deformity than the other lectin complexes. The three lectins interacting with carbohydrates were docked against five individual mutations that frequently occur in major SARS-CoV-2 variants. These were in the spike protein's receptor-binding motif (RBM), while Smo125663 and Smo99732 only interacted with the spike glycoprotein in a protein-protein manner. The precursors for the Hevein-like homologs underwent additional characterization, and their expressional profile in different tissues was studied. These in silico findings offered potential lectin candidates targeting key N-glycan sites crucial to the virus's virulence and infection.

A systematic review on antimicrobial activities of green synthesised Selaginella silver nanoparticles.

BACKGROUND: Metallic nanoparticles from different natural sources exhibit superior therapeutic options as compared to the conventional methods. Selaginella species have attracted special attention of researchers worldwide due to the presence of bioactive molecules such as flavonoids, biflavonoids, triterpenes, steroids, saponins, tannins and other secondary metabolites that exhibit antimicrobial, antiplasmodial, anticancer and anti-inflammatory activities. Environment friendly green synthesised silver nanoparticles from Selaginella species provide viable, safe and efficient treatment against different fungal pathogens. OBJECTIVE: This systematic review aims to summarise the literature pertaining to superior antifungal ability of green synthesised silver nanoparticles using plant extracts of Selaginella spp. in comparison to both aqueous and ethanolic raw plant extracts by electronically collecting articles from databases. METHODS: The recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis were taken into consideration while preparing this review. The titles and abstracts of the collected data were stored in Endnote20 based on the inclusion and exclusion criteria. The search strategy included literature from established sources like PubMed, Google Scholar and Retrieval System Online using subject descriptors. RESULTS: The search yielded 60 articles with unique hits. After removal of duplications, 46 articles were identified, 40 were assessed and only seven articles were chosen and included in this review based on our eligibility criteria. CONCLUSION: The physicochemical and preliminary phytochemical investigations of Selaginella suggest higher drug potency of nanoparticles synthesised from plant extract against different diseases as compared to aqueous and ethanolic plant extracts. The study holds great promise as the synthesis of nanoparticles involves low energy consumption, minimal technology and least toxic effects.

Screening for α-glucosidase inhibitors from Selaginella uncinata based on the ligand fishing combined with ultra-high-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry.

Biflavonoids are naturally occurring compounds consisting of two flavonoid moieties that have received substantial attention from researchers. Although many kinds of biflavonoids are typically distributed in Selaginella uncinata with hypoglycemic effect, their anti-α-glucosidase activities are not yet clear. In this study, a ligand fishing strategy for fast screening of α-glucosidase inhibitors from S. uncinata was proposed. α-Glucosidase was first immobilized on Fe3 O4 magnetic nanoparticles (MNPs) and then the α-glucosidase-functionalized MNPs were incubated with crude extracts of S. uncinata to fish out the ligands. Furthermore, considering the similarity and easy confusion of the structures of biflavonoids, the fragmentation patterns of different types of biflavonoids were studied. Based on this, 11 biflavonoids ligands with α-glucosidase inhibitory activities were accurately and quickly identified from S. uncinata with ultra-high-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry. Furthermore, these ligands were confirmed to be potential inhibitors through the in vitro inhibitory assay and molecular docking.

Phytoconstituents of Selaginella effusa Alston and Their α-Glucosidase as well as Urease Inhibitory Activities.

Three new compounds (1-2, 14), as well as 22 known compounds (3-13, 15-25), were extracted for the first time from the Selaginella effusa Alston (S. effusa). For the unknown compounds, the planar configurations were determined via NMR and by high-resolution mass spectrometry, while their absolute configurations were determined by calculated electronic circular dichroism (ECD), and the configuration of the stereogenic center of biflavones 4-5 were established for the first time. The pure compounds (1-25) were tested in vitro to determine the inhibitory activity of the enzyme-catalyzed reactions. Compounds 1-9 inhibited α-glucosidase with IC50 values ranging from 0.30±0.02 to 4.65±0.04 µM and kinetic analysis of enzyme inhibition indicated that biflavones 1-3 were mixed-type α-glucosidase inhibitors. Compounds 12-13 showed excellent inhibitory activity against urease, with compound 12 (IC50 =4.38±0.31 µM) showing better inhibitory activity than the positive control drug AHA (IC50 13.52±0.61 µM). In addition, molecular docking techniques were used to simulate inhibitor-enzyme binding and to estimate the binding posture of the α-glucosidase and urease catalytic sites.

Three New Selaginellin Derivatives from Selaginella pulvinata and Their α-Glucosidase Inhibitory Activity.

Three new selaginellin derivatives, selaginpulvilins V-X (1-3), together with seven known analogs (4-10) were isolated from whole plants of Selaginella pulvinata. Their structures were determined by extensive spectroscopic methods including 1D and 2D NMR, HR-ESI-MS and chemical derivatization method. Compound 1 represents a rare example of naturally occurring selaginellin with an alkynylphenol-trimmed skeleton. Biological evaluation showed that compounds 2, 6 and 8 displayed moderate inhibition against α-glucosidase with IC50 values of 3.71, 2.04 and 4.00 µM, respectively.

Advances in the Anti-Tumor Activity of Biflavonoids in Selaginella.

Despite the many strategies employed to slow the spread of cancer, the development of new anti-tumor drugs and the minimization of side effects have been major research hotspots in the anti-tumor field. Natural drugs are a huge treasure trove of drug development, and they have been widely used in the clinic as anti-tumor drugs. Selaginella species in the family Selaginellaceae are widely distributed worldwide, and they have been well-documented in clinical practice for the prevention and treatment of cancer. Biflavonoids are the main active ingredients in Selaginella, and they have good biological and anti-tumor activities, which warrant extensive research. The promise of biflavonoids from Selaginella (SFB) in the field of cancer therapy is being realized thanks to new research that offers insights into the multi-targeting therapeutic mechanisms and key signaling pathways. The pharmacological effects of SFB against various cancers in vitro and in vivo are reviewed in this review. In addition, the types and characteristics of biflavonoid structures are described in detail; we also provide a brief summary of the efforts to develop drug delivery systems or combinations to enhance the bioavailability of SFB monomers. In conclusion, SFB species have great potential to be developed as adjuvant or even primary therapeutic agents for cancer, with promising applications.

Three New Benzophenone Derivatives from Selaginella tamariscina.

Six compounds including three new benzophenones, selagibenzophenones D-F (1-3), two known selaginellins (4-5) and one known flavonoid (6), were isolated from Selaginella tamariscina. The structures of new compounds were established by 1D-, 2D-NMR and HR-ESI-MS spectral analyses. Compound 1 represents the second example of diarylbenzophenone from natural sources. Compound 2 possesses an unusual biphenyl-bisbenzophenone structure. Their cytotoxicity against human hepatocellular carcinoma HepG2 and SMCC-7721 cells and inhibitory activities on lipopolysaccharide-induced nitric oxide (NO) production in RAW264.7 cells were evaluated. Compound 2 showed moderate inhibitory activity against HepG2 and SMCC-7721 cells, and compounds 4 and 5 showed moderate inhibitory activity to HepG2 cells. Compounds 2 and 5 also exhibited inhibitory activities on lipopolysaccharide-induced nitric oxide (NO) production.

Vascular function of the T3/modern clade WUSCHEL-Related HOMEOBOX transcription factor genes predate apical meristem-maintenance function.

BACKGROUND: Plants have the lifelong ability to generate new organs due to the persistent functioning of stem cells. In seed plants, groups of stem cells are housed in the shoot apical meristem (SAM), root apical meristem (RAM), and vascular cambium (VC). In ferns, a single shoot stem cell, the apical cell, is located in the SAM, whereas each root initiates from a single shoot-derived root initial. WUSCHEL-RELATED HOMEOBOX (WOX) family transcription factors play important roles to maintain stem-cell identity. WOX genes are grouped phylogenetically into three clades. The T3WOX/modern clade has expanded greatly in angiosperms, with members functioning in multiple meristems and complex developmental programs. The model fern Ceratopteris richardii has only one well-supported T3WOX/modern WOX gene, CrWUL. Its orthologs in Arabidopsis, AtWUS, AtWOX5, and AtWOX4, function in the SAM, RAM, and VC, respectively. Identifying the function of CrWUL will provide insights on the progenitor function and the diversification of the modern WOX genes in seed plants. RESULTS: To investigate the role of CrWUL in the fern, we examined the expression and function of CrWUL and found it expresses during early root development and in vasculature but not in the SAM. Knockdown of CrWUL by RNAi produced plants with fewer roots and fewer phloem cells. When expressed in Arabidopsis cambium, CrWUL was able to complement AtWOX4 function in an atwox4 mutant, suggesting that the WOX function in VC is conserved between ferns and angiosperms. Additionally, the proposed progenitor of T3WOX genes from Selaginella kraussiana is expressed in the vasculature but not in the shoot apical meristem. In contrast to the sporophyte, the expression of CrWUL in the gametophyte exhibits a more general expression pattern and when knocked down, offered little discernable phenotypes. CONCLUSIONS: The results presented here support the occurrence of co-option of the T3WOX/modern clade gene from the gametophyte to function in vasculature and root development in the sporophyte. The function in vasculature is likely to have existed in the progenitor of lycophyte T3WOX/modern clade genes and this function predates its SAM function found in many seed plants.

Plastid phylogenomic analyses of the Selaginella sanguinolenta group (Selaginellaceae) reveal conflict signatures resulting from sequence types, outlier genes, and pervasive RNA editing.

Different from the generally conserved plastomes (plastid genomes) of most land plants, the Selaginellaceae plastomes exhibit dynamic structure, high GC content and high substitution rates. Previous plastome analyses identified strong conflict on several clades in Selaginella, however the factors causing the conflictions and the impact on the phylogenetic inference have not been sufficiently investigated. Here, we dissect the distribution of phylogenetic signals and conflicts in Selaginella sanguinolenta group, the plastome of which is DR (direct repeats) structure and with genome-wide RNA editing. We analyzed the data sets including 22 plastomes representing all species of the S. sanguinolenta group, covering the entire geographical distribution from the Himalayas to Siberia and the Russian Far East regions. We recovered four different topologies by applying multispecies coalescent (ASTRAL) and concatenation methods (IQ-TREE and RAxML) on four data sets of PC (protein-coding genes), NC (non-coding sequences), PCN (the concatenated PC and NC), and RC (predicted RNA editing sites "C" were corrected by "T"), respectively. Six monophyletic clades, S. nummularifolia clade, S. rossii clade, S. sajanensis clade, S. sanguinolenta I clade, S. sanguinolenta II clade, and S. sanguinolenta III clade, were consistently resolved and supported by the characteristics of GC content, RNA editing frequency, and gene content. However, the relationships among these clades varied across the four topologies. To explore the underlying causes of the uncertainty, we compared the phylogenetic signals of the four topologies. We identified that the sequence types (coding versus non-coding), outlier genes (genes with extremely high |ΔGLS| values), and C-to-U RNA editing frequency in the protein-coding genes were responsible for the unstable phylogenomic relationship. We further revealed a significant positive correlation between the |ΔGLS| values and the variation coefficient of the RNA editing number. Our results demonstrated that the coalescent method performed better than the concatenation method in overcoming the problems caused by outlier genes and extreme RNA editing events. Our study particularly focused on the importance of exploring the plastid phylogenomic conflicts and suggested conducting concatenated analyses cautiously when adopting organelle genome data.

Plastome structure, evolution, and phylogeny of Selaginella.

As one of the earliest land plant lineages, Selaginella is important for studying land plant evolution. It is the largest genus of lycophytes containing 700-800 species. Some unique characters of Selaginella plastomes have been reported, but based only on 20 species. There have been no plastome phylogenies of Selaginella based on a relatively large sampling, and no efforts have been made to resolve the phylogeny of the enigmatic Sinensis group whose relationships have been unclear based on small datasets. Here we investigated the structures of 59 plastomes representing 51 species covering all six subgenera and 18 sections of Selaginella except two sections and including the intriguing Sinensis group for the first time. Our major results include: (1) the plastome size of Selaginella ranges tremendously from 78,492 bp to 187,632 bp; (2) there are numerous gene losses in Selaginella comparing with other lycophytes, Isoëtaceae and Lycopodiaceae; (3) the gene contents and plastome structures in Selaginella vary lineage-specifically and all infrageneric taxa are well supported in the plastome phylogeny; (4) the ndh gene family tends to lose or pseudogenize in those species with DR structure and without other short or medium repeats; (5) the short and medium repeat regions in SC mediate many conformations causing diverse and complex plastome structures, and six new conformations are discovered; (6) forty-eight species sampled have high GC content (>50%) but three species in the Sinensis group have âˆ¼ 30% GC content in plastomes, similar to most vascular plants; (7) the Sinensis group is monophyletic, includes at least two subgroups, and has the smallest plastomes in land plants except some parasitic plants, and their plastomes do not contain any tRNAs; (8) the younger lineages in Selaginella tend to have higher GC content, whereas the older lineages tend to have lower GC content; and (9) because of incomplete genomic data and abnormal structures or some unknown reasons, even the concatenated plastomes could not well resolve the phylogenetic relationships in Selaginella with confidence, highlighting the difficulty in resolving the phylogeny and evolution of this particularly important land plant lineage.

Viability markers for determination of desiccation tolerance and critical stages during dehydration in Selaginella species.

While most plants die below a threshold of water content, desiccation-tolerant species display specific responses that allow them to survive extreme dehydration. Some of these responses are activated at critical stages during water loss and could represent the difference between desiccation tolerance (DT) and death. Here, we report the development of a simple and reproducible system to determine DT in Selaginella species. The system is based on exposure of excised tissue to a dehydration agent inside small containers, and subsequent evaluation for tissue viability. We evaluated several methodologies to determine viability upon desiccation including: triphenyltetrazolium chloride (TTC) staining, the quantum efficiency of PSII, antioxidant potential, and relative electrolyte leakage. Our results show that the TTC test is a simple and accurate assay to identify novel desiccation-tolerant Selaginella species, and can also indicate viability in other desiccation-tolerant models (i.e. ferns and mosses). The system we developed is particularly useful to identify critical points during the dehydration process. We found that a desiccation-sensitive Selaginella species shows a change in viability when dehydrated to 40% relative water content, indicating the onset of a critical condition at this water content. Comparative studies at critical stages could provide a better understanding of DT mechanisms and unravel insights into the key responses to survive desiccation.

Methyl jasmonate induces selaginellin accumulation in Selaginella convoluta.

INTRODUCTION: Selaginellins are specialized metabolites and chemotaxonomic markers for Selaginella species. Despite the growing interest in these compounds as a result of their bioactivities, they are accumulated at low levels in the plant. Hence, their isolation and chemical characterization are often difficult, time consuming, and limiting for biological tests. Elicitation with the phytohormone methyl jasmonate (MeJA) could be a strategy to increase the content of selaginellins addressing their low availability problem, that also impairs pharmacological investigations. MATHERIALS AND METHODS: In this study, we examined MeJA elicitation in Selaginella convoluta plants, a medicinal plant found in northeastern Brazil, by treating them with two different concentrations (MeJA: 50 and 100 µM), followed by chemical profiling after 12, 24 and 48 h after application. Samples were harvested and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RESULTS AND DISCUSSCION: MeJA treatment significantly impacted the chemical phenotype. Regarding shoots differences in the time-dependent increased accumulation of all metabolites when plants were subjected to 100 µM MeJA were observed while in roots, most metabolites had their concentrations decreased in a time-dependent fashion at the same conditions. Results support organ, MeJA concentration and time post-treatment dependence of specialized metabolite accumulation, mainly the flavonoids and selaginellins. The amount of Selaginellin G in shoots of MeJA-treated specimens increased in 5.63-fold relative to control. The molecular networking approach allowed for the putative annotation of 64 metabolites, among them, the MeJA treatment followed by targeted metabolome analysis also allowed to annotate seven unprecedented selaginellins. Additionally, the in silico bioactive potential of the annotated selaginellins highlighted targets related to neurodegenerative disorders, antiproliferative, and antiparasitic issues. Taken together, data point out MeJA exposure as a strategy to induce potentially bioactive selaginellins accumulation in S. convoluta, this approach could enable a deep investigation about the metabolic function of these metabolites in the genus as well as regarding pharmacological exploration of the undervalued potential.

Cytotoxic effects of the biflavonoids isolated from Selaginella trichoclada on MCF-7 cells and its potential mechanism.

A new biflavonoid, (2''S)-6''-methyl-2'',3''-dihydroochnaflavone (1), along with two known ochnaflavones (2, 3), four known amentoflavones (4-7) and two known robustaflavones (8, 9) were obtained from the 70% EtOH extract of Selaginella trichoclada. The chemical structures of isolated compounds were elucidated by extensive spectroscopic analyses. Overall, compounds 1-9 displayed moderate cytotoxic effects against human breast cancer MCF-7 cell lines. Among them, compounds 2 and 8 exhibited relatively strong cytotoxic effects against MCF-7 cells with an IC50 value of 7.7 and 6.9 µΜ, respectively. The results of RNA-sequencing and KEGG functional enrichment analysis showed that 8 could induce ferroptosis in MCF-7 cells by down-regulating the expression of ferroptosis-related genes including ACSL4, NOXO1, NOXA1, ACSL5, STEAP3, LPCAT3, ATG7 and TP53. Then 8 could inhibit the expression of ACSL4 proteins through molecule docking analysis, which showed a strong interaction of - 11.89 Kcal/mol binding energy. Those results indicate that 8 could be chemotherapy agents to fight drug resistance in breast cancer by down-regulating the expression level of ACSL4 proteins via ferroptosis, which needs to be further certified in vitro.