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Transcriptome-wide survey divulged a total of 181 ABC transporters in G. glabra which were phylogenetically classified into six subfamilies. Protein–Protein interactions revealed nine putative GgABCBs (-B6, -B14, -B15, -B25, -B26, -B31, -B40, -B42 &-B44) corresponding to five AtABCs orthologs (-B1, -B4, -B11, -B19, &-B21). Significant transcript accumulation of ABCB6 (31.8 folds), - B14 (147.5 folds), - B15 (17 folds), -B25 (19.7 folds), - B26 (18.31 folds), -B31 (61.89 folds), -B40 (1273 folds) and - B42 (51 folds) was observed under the influence of auxin. Auxin transport-specific inhibitor, N-1-naphthylphthalamic acid, showed its effectiveness only at higher (10 µM) concentration where it down regulated the expression of ABCBs , PINs (PIN FORMED) and TWD1 (TWISTED DWARF 1) genes in shoot tissues, while their expression was seen to enhance in the root tissues. Further, qRT-PCR analysis under various growth conditions ( in-vitro , field and growth chamber), and subjected to abiotic stresses revealed differential expression implicating role of ABCBs in stress management. Seven of the nine genes were shown to be involved in the stress physiology of the plant. GgABCB6 , 15 , 25 and ABCB31 were induced in multiple stresses, while GgABCB26 , 40 & 42 were exclusively triggered under drought stress. No study pertaining to the ABC transporters from G. glabra is available till date. The present investigation will give an insight to auxin transportation which has been found to be associated with plant growth architecture; the knowledge will help to understand the association between auxin transportation and plant responses under the influence of various conditions.

The study reports 147 full-length WRKY genes based on the transcriptome analysis of Glycyrrhiza genus ( G. glabra and G. uralensis ). Additional motifs in G. glabra included DivIVA (GgWRKY20) and SerS Superfamily (GgWRKY21) at the C-terminal, and Coat family motifs (GgWRKY55) at the N-terminal of the proteins, while Exo70 exo cyst complex subunit of 338 amino acid (GuWRKY9) was present at the N-terminal of G. uralensis only. Plant Zn cluster super-family domain (17 WRKYs) and bZIP domain (2 WRKYs) were common between the two species. Based on the number of WRKY domains, sequence alignment and phylogenesis, the study identified GuWRKY27 comprising of 3 WRKY domains in G. uralensis and a new subgroup-IIf (10 members), having novel zinc finger pattern (C-X 4 -C-X 22 -HXH) in G. glabra . Multiple WRKY binding domains (1–11) were identified in the promoter regions of the GgWRKY genes indicating strong interacting network between the WRKY proteins. Tissue-specific expression of 25 GgWRKYs , under normal and treated conditions, revealed 11 of the 18 induction factor triggered response corroborating to response observed in AtWRKYs . The study identified auxin-responsive GgWRKY 55 & GgWRKY 38; GA 3 responsive GgWRKYs 15&59 in roots and GgWRKYs 8, 20, 38, 57 &58 in the shoots of the treated plant. GgWRKYs induced under various stresses included GgWRKY 33 (cold), GgWRKY 4 (senescence), GgWRKYs 2, 28 & 33 (salinity) and GgWRKY 40 (wounding). Overall, 23 GgWRKYs responded to abiotic stress, and 17 WRKYs were induced by hormonal signals. Of them 13 WRKYs responded to both suggesting inter-connection between hormone signalling and stress response. The present study will help in understanding the transcriptional reprogramming, protein-protein interaction and cross-regulation during stress and other physiological processes in the plant.

microRNAs represent small endogenous RNAs which are known to play a crucial role in various plant metabolic processes. Carrot being an important vegetable crop, represents one of the richest sources of carotenoids and anthocyanins. Most of the studies on microRNAs have been conducted in the aerial parts of the plants. However, carrot has the rare distinction of storing these compounds in roots. Therefore, carrot represents a good model system to unveil the regulatory roles of miRNAs in the underground edible part of the plant. For the first time, we report the genome wide identification and expression profiling of miRNAs in two contrasting color variants of carrot namely Orange Red and Purple Black using RNA-seq. Illumina sequencing resulted in the generation of 25.5M and 18.9M reads in Orange Red and Purple Black libraries, respectively. In total, 144 and 98 (read count >10), conserved microRNAs and 36 and 66 novel microRNAs were identified in Orange Red and Purple Black, respectively. Functional categorization and differential gene expression revealed the presence of several miRNA genes targeting various secondary metabolic pathways including carotenoid and anthocyanin biosynthetic pathways in the two libraries. 11 known and 2 novel microRNAs were further validated using Stem-Loop PCR and qRT-PCR. Also, target validation was performed for selected miRNA genes using RLM-RACE approach. The present work has laid a foundation towards understanding of various metabolic processes, particularly the color development in carrot. This information can be further employed in targeted gene expression for increasing the carotenoid and anthocyanin content in crop plants.

The study reports a protocol for the regeneration of Glycyrrhiza glabra plantlet from the leaf explant. Notably, the chemical analysis of various developmental stages revealed presence of glycyrrhizin in the underground (7.0–29.8 µg/g) & aerial (7.3–23.4 µg/g) tissues of the in vitro regenerated plants, which was otherwise not detected in the aerial tissues of the field plant. Glycyrrhizin accumulation was reduced or undetected in the regenerated plants transferred to the glasshouse and subsequently under field conditions. Further, spatio-temporal relative gene expression analysis of aerial tissues of in-vitro regenerated G. glabra showed expression of all the known genes committed to glycyrrhizin pathway. In the shoot system, maximum expression of squalene epoxidase (7.9 fold), β-amyrin synthase (21.8 folds), Licorice β-amyrin 11-oxidase (5.9 folds) and UDP-glucosyltransferase (1.7 folds) was observed in different months. However, no expression was detected in the aerial tissues of the field grown plant. Also, a correlation was found between the expression patterns of Licorice β-amyrin 11-oxidase with glycyrrhizin accumulation. This is the first study demonstrating the presence of glycyrrhizin in the aerial tissues of the in-vitro regenerated Glycyrrhiza plant. This study will help in understanding glycyrrhizin regulation, precursor accumulation and their transport. Also, it will unlock the possibilities of understanding and enhancing glycyrrhizin biosynthesis and accumulation in the plant, under in-vitro conditions.

The recent advancements in sequencing technologies and informatic tools promoted a paradigm shift to decipher the hidden biological mysteries and transformed the biological issues into digital data to express both qualitative and quantitative forms. The transcriptomic approach, in particular, has added new dimensions to the versatile essence of plant genomics through the large and deep transcripts generated in the process. This has enabled the mining of super families from the sequenced plants, both model and non-model, understanding their ancestry, diversity, and evolution. The elucidation of the crystal structure of the WRKY proteins and recent advancement in computational prediction through homology modeling and molecular dynamic simulation has provided an insight into the DNA–protein complex formation, stability, and interaction, thereby giving a new dimension in understanding the WRKY regulation. The present review summarizes the functional aspects of the high volume of sequence data of WRKY transcription factors studied from different species, till date. The review focuses on the dynamics of structural classification and lineage in light of the recent information. Additionally, a comparative analysis approach was incorporated to understand the functions of the identified WRKY transcription factors subjected to abiotic (heat, cold, salinity, senescence, dark, wounding, UV, and carbon starvation) stresses as revealed through various sets of studies on different plant species. The review will be instrumental in understanding the events of evolution and the importance of WRKY TFs under the threat of climate change, considering the new scientific evidences to propose a fresh perspective.

Main conclusion ABCs transport diverse compounds; with plant’s most abundant ABCG and ABCB subfamilies. ABCBs are multi-functional transporter proteins having role in plant adaptation. ATP-binding cassette (ABC) proteins have been known for the transportation of various structurally diverse compounds in all kingdoms of life. Plants possess a particularly high number of ABC transporters compared to other eukaryotes: the most abundant being ABCG followed by the ABCB subfamilies. While members of the ABCB subfamily are primarily known for auxin transportation, however, studies have shown their involvement in variety of other functions viz . growth and development, biotic and abiotic stresses, metal toxicity and homeostasis, cellular redox state stability, stomatal regulation, cell shape maintenance, and transport of secondary metabolites and phytohormones. These proteins are able to perform various biological processes due to their widespread localization in the plasma membrane, mitochondrial membrane, chloroplast, and tonoplast facilitating membrane transport influenced by various environmental and biological cues. The current review compiles published insights into the role of ABCB transporters, and also provides brief insights into the role of ABCB transporters in a medicinal plant, where the synthesis of its bioactive secondary metabolite is linked to the primary function of ABCBs, i.e., auxin transport. The review discusses ABCB subfamily members as multi-functional protein and comprehensively examines their role in various biological processes that help plants to survive under unfavorable environmental conditions. Graphical abstract

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.

Dioscorea deltoidea (Family: Dioscoreaceae) is a critically endangered medicinal plant used in the pharmaceutical industry for the production of steroidal drugs, including cortisone, contraceptives, and sex hormones. This study explored the effects of various elicitors—salicylic acid (SA), methyl jasmonate (MeJa), jasmonic acid (JA), and abscisic acid (ABA), along with precursors (squalene, β-sitosterol, and cholesterol) on diosgenin synthesis in D. deltoidea shoot cultures. The results demonstrated that precursors significantly enhanced diosgenin production compared to elicitors. SA (200 µM) resulted in the highest diosgenin content (0.912% DW) after 4 h of exposure, while MeJa (100 µM) produced 0.814% DW after 8 h. Among the precursors, β-sitosterol (200 µM) yielded the highest diosgenin content (1.006% DW), followed by squalene (100 µM) at 0.947% DW after 5 days. Cholesterol showed lower diosgenin production, though it was still higher than in control cultures. Response Surface Methodology (RSM) was applied to determine the optimal concentrations of elicitors and precursors, revealing that the combination of 165 µM SA and 150 µM β-sitosterol resulted in the highest diosgenin yield (1.321% DW) and biomass production (1.958 g). These findings highlight the significant influence of elicitors and precursors on diosgenin accumulation, offering valuable insights for optimizing its industrial production. Graphical Abstract Highlights Investigation of the effects of salicylic acid (SA), methyl jasmonate (MeJa), jasmonic acid (JA), and abscisic acid (ABA) on diosgenin synthesis in Dioscorea deltoidea shoot cultures. β-Sitosterol was identified as the most effective precursor, yielding 1.006% diosgenin at 200 µM, followed by squalene at 100 µM Salicylic acid (200 µM) produced the highest diosgenin content (0.912% DW) after 4 hours of exposure, with methyl jasmonate (100 µM) showing 0.814% diosgenin after 8 hours. Response surface methodology (RSM) identified optimal conditions for diosgenin and biomass production: 165 µM SA and 150 µM β-sitosterol, resulting in a diosgenin yield of 1.321% and biomass yield of 1.958 g.

Fritillaria roylei is an endangered bulbous perennial plant with high ethnopharmacological value. The high morphophysiological dormancy, habitat destruction, and irresponsive collection have resulted in the endangered status of F. roylei plants. The present study established an efficient in vitro regeneration and multiplication protocol for bulb multiplication. Gamborg’s B5 medium supplemented with NAA (5.3 µM), BAP (4.4 µM) and kinetin (4.6 µM), spermidine (137 µM), and NLN vitamin mixture (1 mg/mL) was used efficiently for bulb multiplication. Each bulb produced 10–12 bulblets after 12–14 wk showing clonal fidelity under standardized in vitro conditions. The phytochemical analysis of the regenerated bulbs detected four important steroidal alkaloids, namely, solanidine, peimisine, petisidine, and fritilline A. Elicitation (jasmonic acid, methyl jasmonate, salicylic acid, and gibberellic acid) of regenerated bulbs using response surface methodology demonstrated higher accumulation of solanidine (2.6 folds), and peimisine, petisidine and fritilline A (up to 1.4 folds each). The study will facilitate mass multiplication and conservation of endangered species of medicinal importance and enable understanding the biosynthesis of bioactives in the species.

Squalene epoxidase (SQE) is a crucial regulatory enzyme for the biosynthesis of several important classes of compounds including sterols and triterpenoids. The present paper identified and characterised five SQE genes (GgSQE1 to GgSQE5) from Glycyrrhiza glabra through transcriptome data mining and homology-based cloning, for the first time. The phylogenetic analysis implied their functional divergence. The ORF corresponding to one of the five SQEs, namely, GgSQE1, was cloned and studied for its function in a heterologous system, following transient and stable expressions. The transient expression followed by GgSQE1 encoding protein purification suggested approximately 58.0-kDa protein following the predicted molecular mass of the deduced protein. The gene expression profiling based on qRT-PCR indicated its highest expression (6.4-folds) in the 10-month-old roots. Furthermore, ABA (12.4-folds) and GA3 (2.47) treatments upregulated the expression of GgSQE1 in the shoots after 10 and 12 hours, respectively, which was also reflected in glycyrrhizin accumulation. The inductive effects of ABA and GA3 over GgSQE1 expression were also confirmed through functional analysis of GgSQE1 promoters using GUS fusion construct. Stable constitutive expression of GgSQE1 in Nicotiana tabacum modulated the sterol contents. The study could pave the way for understanding the metabolic flux regulation concerning biosynthesis of related sterols and triterpenoids.