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Plumbago auriculata L., a tropical evergreen, developed enhanced cold tolerance through long-term field acclimation, yet its molecular mechanisms remained unclear. Here, we identified PaMYBS3 , a MYB transcription factor, as a key dual-function regulator during cold acclimation. Short-term artificial cold treatment induced PaMYBS3 expression and improved cold resistance. Overexpression of PaMYBS3 in Arabidopsis promoted growth but reduced freezing tolerance, while domain-mutant and VIGS-silenced lines exhibited inhibited growth and enhanced cold adaptation. Physiological assays revealed that PaMYBS3 enhances cold tolerance by modulating ROS balance, photosynthetic performance, and osmotic regulators, while suppressing growth through downregulation of auxin- and sugar-related genes, supporting a cold acclimation strategy that prioritizes survival over growth. Notably, PaMYBS3 regulated cold-responsive pathways independently of the classical CBF/DREB1-COR module, suggesting a CBF-independent cold tolerance mechanism. Our findings highlight PaMYBS3 as a central integrator of cold response and growth restraint, offering new insights into the adaptive strategies of tropical perennials under prolonged low temperature.

Main conclusionEnhanced secretion of Na+ and Cl− in leaf glands and leaf vacuolar sequestration of Na+ or root retention of Cl−, combined with K+ retention, contribute to the improved salt tolerance of tetraploid recretohalophyte P. auriculata.Salt stress is one of the major abiotic factors threatening plant growth and development, and polyploids generally exhibit higher salt stress resistance than diploids. In recretohalophytes, which secrete ions from the salt gland in leaf epidermal cells, the effects of polyploidization on ion homeostasis and secretion remain unknown. In this study, we compared the morphology, physiology, and ion homeostasis regulation of diploid and autotetraploid accessions of the recretohalophyte Plumbago auriculata Lam. after treatment with 300 mM NaCl for 0, 2, 4, 6, and 8 days. The results showed that salt stress altered the morphology, photosynthetic efficiency, and chloroplast structure of diploid P. auriculata to a greater extent than those of its tetraploid counterpart. Moreover, the contents of organic osmoregulatory substances (proline and soluble sugars) were significantly higher in the tetraploid than in the diploid, while those of H2O2 and malondialdehyde (MDA) were significantly lower. Analysis of ion homeostasis revealed that the tetraploid cytotype accumulated more Na+ in stems and leaves and more Cl− in roots but less K+ loss in roots compared with diploid P. auriculata. Additionally, the rate of Na+ and Cl− secretion from the leaf surface was higher, while that of K+, Mg2+, and Ca2+ secretion was lower in tetraploid plants. X-ray microanalysis of mesophyll cells revealed that Na+ mainly accumulated in different cellular compartments in the tetraploid (vacuole) and diploid (cytoplasm) plants. Our results suggest that polyploid recretohalophytes require the ability to sequester Na+ and Cl−(via accumulation in leaf cell vacuoles or unloading by roots) and selectively secrete these ions (through salt glands) together with the ability to prevent K+ loss (by roots). This mechanism required to maintain K+/Na+ homeostasis in polyploid recretohalophytes under high salinity provides new insights in the improved maintenance of ion homeostasis in polyploids under salt stress.

Naturally synthesized secondary metabolites in plants are considered an important source of drugs, food additives, etc. Among them, research on natural plant medicinal components and their synthesis mechanisms has always been of high concern. We identified a novel medicinal floral crop, Plumbago auriculata L., that can be treated with methyl jasmonate (MeJA) for the rapid or sustainable production of natural bioactives from hairy roots. In the study, we globally analyzed the changes in the accumulation of plumbagin and others in the hairy roots of Plumbago auriculata L. hairy roots (PAHR) 15834 in P. auriculata L. based on 100 μmol/L of MeJA treatment by RNA-seq profiling, and we found that there was a significant increase in the accumulation of plumbagin and saponin before 24 h. To explain the principle of co-accumulation, it showed that MeJA induced JA signaling and the shikimic acid pathway, and the methylvaleric acid (MVA) pathway was activated downstream subsequently by the Mfuzz and weighted gene co-expression analysis. Under the shared metabolic pathway, the high expression of PAL3 and HMGR promoted the activity of the “gateway enzymes” phenylalanine ammonia lyase (PAL) and 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), which respectively induced the high expression of key reaction enzyme genes, including chalcone synthase (CHS), isopentenyl diphosphate (IPP), and farnesyl pyrophosphate synthase (FPS), that led to the synthesis of plumbagin and saponin. We speculated that large amounts of ketones and/or aldehydes were formed under the action of these characteristic enzymes, ultimately achieving their co-accumulation through polyketone and high-level sugar and amino acid metabolism. The study results provided a theoretical basis for carrying out the factory refinement and biosynthesis of plumbagin and saponins and also provided new ideas for fully exploiting multifunctional agricultural crops and plants and developing new agricultural by-products.

Zinc oxide nanoparticles (ZnO NPs) were synthesized by using an alcoholic extract of the flowering aerial parts of Plumbago auriculata Lam. Dynamic Light Scattering (DLS) revealed that the average size of synthesized ZnO NPs was 10.58 ± 3.350 nm and the zeta potential was −19.6 mV. Transmission electron microscopy (TEM) revealed that the particle size was in the range from 5.08 to 6.56 nm. X-ray diffraction (XRD) analysis verified the existence of pure hexagonal shaped crystals of ZnO nanoparticles with an average size of 35.34 nm in the sample, which is similar to the particle size analysis acquired by scanning electron microscopy (SEM) (38.29 ± 6.88 nm). HPLC analysis of the phenolic ingredients present in the plant extract showed that gallic acid, chlorogenic acid, and catechin were found as major compounds at concentrations of 1720.26, 1600.42, and 840.20 µg/g, respectively. Furthermore, the inhibitory effects of ZnO NPs and the plant extract against avian metapneumovirus (aMPV) subtype B were also investigated. This assessment revealed that the uncalcinated form of Nano-ZnO mediated by P. auriculata Lam. extract possessed a significant antiviral activity with 50% cytotoxic concentration (CC50) and 50% inhibition concentration (IC50) of 52.48 ± 1.57 and 42.67 ± 4.08 µg/mL, respectively, while the inhibition percentage (IP) was 99% and the selectivity index (SI) was 1.23.

This study aimed at investigating the chemical composition and the hepatoprotective activities of Plumbago indica L. and P. auriculata Lam . LC–MS/MS analyses for the hydroalcoholic extracts of the aerial parts of the two Plumbago species allowed the tentative identification of thirty and twenty-five compounds from P. indica and P. auriculata , respectively. The biochemical and histopathological alterations associated with thioacetamide (TAA)-induced liver fibrosis in rats were evaluated in vivo where rats received the two extracts at three different dose levels (100, 200 and 400 mg/kg p.o, daily) for 15 consecutive days with induction of hepatotoxicity by TAA (200 mg/kg/day, i.p.) at 14th and 15th days. Results of the present study showed a significant restoration in liver function biomarkers viz . alanine transaminase (ALT), aspartate transaminase (AST), gamma glutamyl transferase and total bilirubin. The liver homogenates exhibited increased levels of antioxidant biomarkers: reduced glutathione (GSH) and catalase (CAT), accompanied with decline in malondialdehyde (MDA). Furthermore, treated groups exhibited a significant suppression in liver inflammatory cytokines: tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6), and fibrotic biomarker: alpha smooth muscle relaxant. Histopathological examination of the liver showed normality of hepatocytes. Noteworthy, P. indica extract showed better hepatoprotective activity than P. auriculata , particularly at 200 mg/kg. To sum up, all these results indicated the hepatoprotective properties of both extracts, as well as their antifibrotic effect was evidenced by reduction in hepatic collagen deposition. However, additional experiments are required to isolate their individual secondary metabolites, assess the toxicity of the extracts and explore the involved mechanism of action.

A new spodumene-rich pegmatite was recently discovered on the north side of Plumbago Mountain, Oxford County, Western Maine. The site is about 1.5 km northwest of the famous Dunton gem tourmaline pegmatite. It is an albite-quartz-spodumene pegmatite containing gigantic crystals of spodumene and montebrasite. Some spodumene crystals are more than 11 m in length. The upper portions of the pegmatite contain up to 50 wt% spodumene, which makes it a potentially significant new lithium resource. Preliminary results indicate that Plumbago North Pegmatite contains about 10 Mt of ore with an average Li2O content of 4.68 wt% which is a higher average lithium content than any of the ten top spodumene-producing deposits in the world.

The genus Plumbago (family Plumbaginaceae), commonly known as leadwort, is a sub-tropical shrub that produces secondary metabolite plumbagin, which is employed by pharmaceutical companies and in clinical research. Plumbagin is a potent pharmaceutical because of its anti-microbial, anti-malarial, antifungal, anti-inflammatory, anti-carcinogenic, anti-fertility, anti-plasmodium, antioxidant, anti-diabetic, and other effects. This review documents the biotechnological innovations used to produce plumbagin. The use of modern biotechnological techniques can lead to a variety of benefits, including better yield, increased extraction efficiency, mass production of plantlets, genetic stability, increased biomass, and more. Large-scale in vitro propagation is necessary to minimize over-exploitation of the natural population and allow the use of various biotechnological techniques to improve the plant species and secondary metabolite production. During in vitro culture, optimum conditions are requisites for explant inoculation and plant regeneration. In this review, we provide information on various aspects of plumbagin, depicting its structure, biosynthesis, and biotechnological aspects (both conventional and advanced) along with the future prospects.Key Points• Critical assessment on in vitro biotechnology in Plumbago species• In vitro propagation of Plumbago and elicitation of plumbagin• Biosynthesis and sustainable production of plumbagin

Plumbagin is the major secondary metabolite found in Plumbago species, it exhibit a wide variety of pharmaceutical activities. The initial focus of this study was to establish a suitable protocol for direct shoot regeneration from nodal explants on Murashige and Skoog (MS) medium fortified with 6-benzylaminopurine (BAP) and α-naphthalene-acetic acid (NAA). Single shoot regenerated from the nodal explants when cultured on BAP alone, whereas multiple shoots regenerated with BAP and NAA. The maximum number of shoots were obtained on medium fortified with 5.5 µM BAP and 3.0 µM NAA. Furthermore, the shoot cultures were treated with biotic elicitors (lysates of Agrobacterium rhizogenes and Trichoderma viride and yeast extract). The application of elicitors was optimized by the Response Surface Methodology coupled with Box-Behnken design (RSM-BBD). The experimental and predicted values were analyzed and the elicitors were found to be stastically significant. Plumbagin was quantified by liquid chromatography/electrospray ionization tandem. The highest plumbagin yield was found to be 18-fold higher as compared to the control when treated with 1.5% A. rhizogenes lysate, 2.25% T. viride lysate and 2% yeast extract. The best model for optimum production of plumbagin was experimentally verified and the correlation between the predicted (1.751%) and actual values (1.732%) was found to be 98.7% for plumbagin production. The present study suggest that the elicitors used enhance the production of plumbagin considerably. It is the first ever study to report the successful utilization of lysates enhancing plumbagin production in the shoot cultures.Key messageIn vitro production of plumbagin from Plumbago zeylenica. Analysis of plumbagin by LC-MS. Application of RSM to optimize the enhanced plumbagin yield by using eliciters.

Plumbaginaceae is characterized by a history of multiple taxonomic rearrangements and lacks a broad molecular phylogenetic framework. Limonium is the most speciesrich genus of the family with ca. 600 species and cosmopolitan distribution. Its center of diversity is the Mediterranean region, where ca. 70% of all Limonium species are endemic. In this study, we sample 201 Limonium species covering all described infrageneric entities and spanning its wide geographic range, along with 64 species of other Plumbaginaceae genera, representing 23 out of 29 genera of the family. Additionally, 20 species of the sister family Polygonaceae were used as outgroup. Sequences of three chloroplast (trnL‐F, matK, and rbcL) and one nuclear (ITS) loci were used to infer the molecular phylogeny employing maximum likelihood and Bayesian analyses. According to our results, within Plumbaginoideae, Plumbago forms a nonmonophyletic assemblage, with Plumbago europaea sister to Plumbagella, while the other Plumbago species form a clade sister to Dyerophytum. Within Limonioideae, Ikonnikovia is nested in Goniolimon, rejecting its former segregation as genus distinct from Goniolimon. Limonium is divided into two major clades: Limonium subg. Pteroclados s.l., including L. sect. Pteroclados and L. anthericoides, and L. subg. Limonium. The latter is divided into three well‐supported subclades: the monospecific L. sect. Limoniodendron sister to a clade comprising a mostly non‐Mediterranean subclade and a Mediterranean subclade. Our results set the foundation for taxonomic proposals on sections and subsections of Limonium, namely: (a) the newly described L. sect. Tenuiramosum, created to assign L. anthericoides at the sectional rank; (b) the more restricted circumscriptions of L. sect. Limonium (= L. sect. Limonium subsect. Genuinae) and L. sect. Sarcophyllum (for the Sudano‐Zambezian/Saharo‐Arabian clade); (c) the more expanded circumscription of L. sect. Nephrophyllum (including species of the L. bellidifolium complex); and (d) the new combinations for L. sect. Pruinosum and L. sect. Pteroclados subsect. Odontolepideae and subsect. Nobiles.

Under salt stress, recretohalophyte Plumbago auriculata tetraploids enhance salt tolerance by increasing selective secretion of Na + compared with that in diploids, although the mechanism is unclear. Using non-invasive micro-test technology, the effect of salt gland Ca 2+ content on Na + and K + secretion were investigated in diploid and tetraploid P. auriculata under salt stress. Salt gland Ca 2+ content and secretion rates of Na + and K + were higher in tetraploids than in diploids under salt stress. Addition of exogenous Ca 2+ increased the Ca 2+ content of the salt gland in diploids and is accompanied by an increase in the rate of Na + and K + secretion. With addition of a Ca 2+ channel inhibitor, diploid salt glands retained large amounts of Ca 2+ , leading to higher Ca 2+ content and Na + secretion rate than those of tetraploids. Inhibiting H 2 O 2 generation and H + -ATPase activity altered Na + and K + secretion rates in diploids and tetraploids under salt stress, indicating involvement in regulating Na + and K + secretion. Our results indicate that the increased Na + secretion rate of salt gland in tetraploids under salt stress was associated with elevated Ca 2+ content in salt gland.