Genome-wide identification and characterization of wall-associated kinases, molecular docking and polysaccharide elicitation of monoterpenoid indole alkaloids in micro-propagated Catharanthus roseus.

Wall-associated kinases (WAKs) are a unique family of proteins that are predominantly localized on the plasma membrane and simultaneously bound to the cell wall. WAKs play a pivotal role in signal transduction to regulate growth, defense, and response to environmental stimuli in plants. These kinases have been identified and characterized in various plant species, however, similar information for Catharanthus roseus is scarce. C. roseus is an evergreen ornamental plant that produces a repertoire of biologically active compounds. The plant is best characterized for the production of antineoplastic monoterpenoid indole alkaloids (MIAs) namely vinblastine and vincristine. Owing to the diverse composition of phytochemicals, C. roseus is known as a "model non-model" plant for secondary metabolite research. Genome analyses showed 37 putative CrWAK genes present in C. roseus, largely localized on the plasma membrane. Phylogenetic analysis revealed six clusters of CrWAKs. Diverse cis-acting elements, including those involved in defense responses, were identified on the promotor regions of CrWAK genes. The highest binding affinity (- 12.6 kcal/mol) was noted for CrWAK-22 against tri-galacturonic acid. Tri-galacturonic acid stimulated 2.5-fold higher production of vinblastine, sixfold upregulation of the expression of ORCA3 transcription factor, and 6.14-fold upregulation of CrWAK-22 expression. Based on these results it was concluded that the expression of CrWAK genes induced by biotic elicitors may have an important role in the production of MIAs. The current findings may serve as a basis for functional characterization and mechanistic explanation of the role of CrWAK genes in the biosynthesis of MIAs upon elicitation.

Real-time expression and in silico characterization of pea genes involved in salt and water-deficit stress.

BACKGROUND: To tolerate salt and water-deficit stress, the plant adapts to the adverse environment by regulating its metabolism and expressing certain stress-induced metabolic pathways. This research analyzed the relative expression of four pea genes (P5CR, PAL1, SOD, and POX) in three pea varieties (Climax, Green grass, and Meteor) under different levels of salt and water-deficit stress. METHODS AND RESULTS: The experiments on salt stress and water-deficit stress were carried out within greenhouse settings under controlled environment. The saturation percentage was employed to create artificial salinity conditions: Control without NaCl treatment, Treatment 1: 50 mM NaCl treatment, Treatment 2: 75 mM NaCl treatment, and Treatment 3: 100 mM NaCl treatment. Field capacity (FC) was used for the development of artificial water-deficit treatments in the pots, i.e., Treatment 1 (Control; water application 100% of FC), Treatment 2 (water application 75% of FC), and Treatment 3 (water application 50% of FC). Pea genes involved in biosynthetic pathways of proline, flavonoids, and enzymatic antioxidant enzymes including P5CR, PAL1, SOD, and POX were selected based on literature. Quantitative real-time PCR using cDNA as a template was used to analyze the gene expression. Pea genes were analyzed for phylogenetic analysis in closely related crops having similarity percent identity 80% and above. In silico characterization of selected proteins including the family classification was done by the NCBI CDD and INTERPRO online servers. Results from RT-qPCR analysis showed increased expression of P5CR, PAL1, and POX genes, while SOD gene expression decreased under both stresses. Climax exhibited superior stress tolerance with elevated expression of P5CR and PAL1, while Meteor showed better tolerance through increased POX expression. Phylogenetic analysis revealed common ancestry with other species like chickpea, red clover, mung bean, and barrel clover, suggesting the cross relationship among these plant species. Conserved domain analysis of respective proteins revealed that these proteins contain PLNO 2688, PLN02457, Cu-Zn Superoxide dismutase, and secretory peroxidase conserved domains. Furthermore, protein family classification indicated that the oxidation-reduction process is the most common chemical process involved in these stresses given to pea plant which validates the relationship of these proteins. CONCLUSIONS: Salt and water-deficit stresses trigger distinct metabolic pathways, leading to the up-regulation of specific genes and the synthesis of corresponding proteins. These findings further emphasize the conservation of stress-tolerance-related genes and proteins across various plant species. This knowledge enhances our understanding of plant adaptation to stress and offers opportunities for developing strategies to improve stress resilience in crops, thereby addressing global food security challenges.

Evaluating the antioxidative defense response of selected indoor plants against benzene and formaldehyde.

Volatile organic compounds (VOCs) such as formaldehyde and benzene are among the key contributors to indoor air pollution. The current situation of environmental pollution is alarming, especially indoor air pollution is becoming a challenge as affecting plants and humans. VOCs are known to adversely affect indoor plants by causing necrosis and chlorosis. In order to withstand these organic pollutants, plants are naturally equipped with an antioxidative defense system. The current research study aimed to evaluate the combined effect of formaldehyde and benzene on the antioxidative response of selected indoor C3 plants including Chlorophytum comosum, Dracaena mysore, and Ficus longifolia. After the combined application of different levels (0, 0; 2, 2; 2, 4; 4, 2; and 4, 4 ppm) of benzene and formaldehyde respectively, in an airtight glass chamber, the enzymatic and non-enzymatic antioxidants were analyzed. Analysis of total phenolics showed a significant increase (10.72 mg GAE/g) in F. longifolia; C. comosum (9.20 mg GAE/g); and D. mysore (8.74 mg GAE/g) compared to their respective controls as 3.76, 5.39, and 6.07 mg GAE/g. Total flavonoids (724 µg/g) were reported in control plants of F. longifolia which were increased to 1545.72 µg/g from 724 µg/g (in control) followed by 322.66 µg/g in D. mysore (control having 167.11 µg/g). Total carotenoid content also increased in D. mysore (0.67 mg/g) followed by C. comosum (0.63 mg/g) in response to increasing the combined dose compared to their control plants having 0.62 and 0.24 mg/g content. The highest proline content was exhibited by D. mysore (3.66 µg/g) as compared to its respective control plant (1.54 µg/g) under a 4 ppm dose of benzene and formaldehyde. A significant increase in enzymatic antioxidants including total antioxidants (87.89%), catalase (59.21 U/mg of protein), and guaiacol peroxidase (52.16 U/mg of protein) was observed in the D. mysore plant under a combined dose of benzene (2 ppm) and formaldehyde (4 ppm) with respect to their controls. Although experimental indoor plants have been reported to metabolize indoor pollutants, the current findings indicate that the combined application of benzene and formaldehyde is also affecting the physiology of indoor plants as well.

Genome-wide identification of NAC transcription factors and regulation of monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus.

NAC transcription factors (TFs) are crucial to growth and defense responses in plants. Though NACs have been characterized for their role in several plants, comprehensive information regarding their role in Catharanthus roseus, a perennial ornamental plant, is lacking. Homology modelling was employed to identify and characterize NACs in C. roseus. In-vitro propagation of C. roseus plants was carried out using cell suspension and nodal culture and were elicited with two auxin-antagonists, 5-fluoro Indole Acetic Acid (5-F-IAA) and α-(phenyl ethyl-2-oxo)-Indole-Acetic-Acid (PEO-IAA) for the enhanced production of monoterpenoid indole alkaloids (MIAs) namely catharanthine, vindoline, and vinblastine. Analyses revealed the presence of 47 putative CrNAC genes in the C. roseus genome, primarily localized in the nucleus. Phylogenetic analysis categorized these CrNACs into eight clusters, demonstrating the highest synteny with corresponding genes in Camptotheca acuminata. Additionally, at least one defense or hormone-responsive cis-acting element was identified in the promoter region of all the putative CrNACs. Of the two elicitors, 5-F-IAA was effective at 200 µM to elicit a 3.07-fold increase in catharanthine, 2.76-fold in vindoline, and 2.4-fold in vinblastine production in nodal culture. While a relatively lower increase in MIAs was recorded in suspension culture. Validation of RNA-Seq by qRT-PCR showed upregulated expression of stress-related genes (CrNAC-07 and CrNAC-24), and downregulated expression of growth-related gene (CrNAC-25) in elicited nodal culture of C. roseus. Additionally, the expression of genes involved in the biosynthesis of MIAs was significantly upregulated upon elicitation. The current study provides the first report on the role of CrNACs in regulating the biosynthesis of MIAs.

The Interplay between Toxic and Essential Metals for Their Uptake and Translocation Is Likely Governed by DNA Methylation and Histone Deacetylation in Maize.

The persistent nature of lead (Pb) and cadmium (Cd) in the environment severely affects plant growth and yield. Conversely, plants acquire zinc (Zn) from the soil for their vital physiological and biochemical functions. However, the interplay and coordination between essential and toxic metals for their uptake and translocation and the putative underlying epigenetic mechanisms have not yet been investigated in maize. Here, we report that the presence of Zn facilitates the accumulation and transport of Pb and Cd in the aerial parts of the maize plants. Moreover, the Zn, Pb, and Cd interplay specifically interferes with the uptake and translocation of other divalent metals, such as calcium and magnesium. Zn, Pb, and Cd, individually and in combinations, differentially regulate the expression of DNA methyltransferases, thus alter the DNA methylation levels at the promoter of Zinc-regulated transporters, Iron-regulated transporter-like Protein (ZIP) genes to regulate their expression. Furthermore, the expression of histone deacetylases (HDACs) varies greatly in response to individual and combined metals, and HDACs expression showed a negative correlation with ZIP transporters. Our study highlights the implication of DNA methylation and histone acetylation in regulating the metal stress tolerance dynamics through Zn transporters and warns against the excessive use of Zn fertilizers in metal contaminated soils.

Physical and chemical mutagens improved Sporotrichum thermophile, strain ST20 for enhanced Phytase activity.

OBJECTIVE: Phosphorous is an essential micronutrient of plants and involved in critical biological functions. In nature, phosphorous is mostly present in immobilized inorganic mineral and in the fixed organic form including phytic acid and phosphoesteric compounds. However, the bioavailability of bound phosphorous could be enhanced by the use of phosphate solubilizing microorganisms such as bacteria and fungi. The phytases are widespread in an environment and have been isolated from different sources comprising bacteria and fungi. METHODOLOGY: In current studies, we show the successful use of gamma rays and EMS (Ethyl Methane Sulphonate) mutagenesis for enhanced activity of phytases in a fungal strain Sporotrichum thermophile. RESULTS: We report an improved strain ST2 that could produce a clear halo zone around the colony, up to 24 mm. The maximum enzymatic activity was found of 382 U/mL on pH 5.5. However, the phytase activity was improved to 387 U/ml at 45 °C. We also report that the mutants produced through EMS showed the greater potential for phytase production. CONCLUSION: The current study highlights the potential of EMS mutagenesis for strain improvement over physical mutagens.

Lead, Cadmium and Zinc Phytotoxicity Alter DNA Methylation Levels to Confer Heavy Metal Tolerance in Wheat.

Being a staple food, wheat (Triticum aestivum) nutritionally fulfills all requirements of human health and also serves as a significant link in the food chain for the ingestion of pollutants by humans and animals. Therefore, the presence of the heavy metals such as lead (Pb) and cadmium (Cd) in soil is not only responsible for the reduction of wheat crop yield but also the potential threat for human and animal health. However, the link between DNA methylation and heavy metal stress tolerance in wheat has not been investigated yet. In this study, eight high yielding wheat varieties were screened based on their phenotype in response to Pb stress. Out of these, Pirsabak 2004 and Fakhar-e-sarhad were identified as Pb resistant and sensitive varieties, respectively. In addition, Pirsabak 2004 and Fakhar-e-sarhad varieties were also found resistant and sensitive to Cd and Zinc (Zn) stress, respectively. Antioxidant activity was decreased in Fakhar-e-sarhad compared with control in response to Pb/Cd/Zn stresses, but Fakhar-e-sarhad and Pirsabak 2004 accumulated similar levels of Pb, Cd and Zn in their roots. The expression of Heavy Metal ATPase 2 (TaHMA2) and ATP-Binding Cassette (TaABCC2/3/4) metal detoxification transporters are significantly upregulated in Pirsabak 2004 compared with Fakhar-e-sarhad and non-treated controls in response to Pb, Cd and Zn metal stresses. Consistent with upregulation of metal detoxification transporters, CG DNA hypomethylation was also found at the promoter region of these transporters in Pirsabak 2004 compared with Fakhar-e-sarhad and non-treated control, which indicates that DNA methylation regulates the expression of metal detoxification transporters to confer resistance against metal toxicity in wheat. This study recommends the farmers to cultivate Pirsabak 2004 variety in metal contaminated soils and also highlights that DNA methylation is associated with metal stress tolerance in wheat.

Effect of Antioxidants, Amino Acids and Plant Growth Regulators on in vitro Propagation of Rosa centifolia.

BACKGROUND: Rosa centifoliais commercially propagated by asexual means but in vitro propagation ensure the production of disease free and healthy plants and browning of explants creates hurdle in their multiplication. OBJECTIVES: The aim was to reduce oxidative browning of shoots of R. centifolia in MS medium during in vitro propagation. MATERIALS AND METHODS: Axillary buds of R. centifolia were sterilized with 70% ethyl alcohol for 4 min and 5% sodium hypochlorite for 2 min followed by three washing with sterilized double distilled water. In order to control oxidative browning, Ascorbic acid (100 mg.L-1), citric acid (100 mg.L-1) and activated charcoal (3 g.L-1) were used while to control withering of shoots, different concentrations (3.0 mg.L-1, 6.0 mg.L-1, 9.0 mg.L-1) of either glutamine, asparagine and proline were put into trial. Different concentrations of Benzyl aminopurine (BAP) and naphthalene acetic acid (NAA) were used for in vitro shoot and root formation. RESULTS: Minimum browning percentage (20%) was achieved in the presence of activated charcoal (3.0 g.L-1) and pretreatment of explants with running tap water. Asparagin (9.0 mg.L-1) produced maximum shooting (93%), minimum withering (6.67%), and it took longer period (27 days) for shoots to wither. BAP (3.0 mg.L-1) + NAA (0.5 mg.L-1) was produced the highest number of shoots (1.63), in a shortest periods (9 days). For root production, NAA (1.5 mg.L-1) + BAP (0.5 mg.L-1) reduced the time to 11 days with maximum number of roots (4.33) and root length (4.20 cm). CONCLUSIONS: The supplement of activated charcoal (3.0 g.L-1), a sparagin (9.0 mg.L-1) and combination of BAP and NAA in the MS medium is effective for in vitro propagation of R. centifolia.

Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination.

Sunflower (Helianthus annuus L.) seed dormancy is regulated by reactive oxygen species (ROS) and can be alleviated by incubating dormant embryos in the presence of methylviologen (MV), a ROS-generating compound. Ethylene alleviates sunflower seed dormancy whereas abscisic acid (ABA) represses germination. The purposes of this study were to identify the molecular basis of ROS effect on seed germination and to investigate their possible relationship with hormone signalling pathways. Ethylene treatment provoked ROS generation in embryonic axis whereas ABA had no effect on their production. The beneficial effect of ethylene on germination was lowered in the presence of antioxidant compounds, and MV suppressed the inhibitory effect of ABA. MV treatment did not alter significantly ethylene nor ABA production during seed imbibition. Microarray analysis showed that MV treatment triggered differential expression of 120 probe sets (59 more abundant and 61 less abundant genes), and most of the identified transcripts were related to cell signalling components. Many transcripts less represented in MV-treated seeds were involved in ABA signalling, thus suggesting an interaction between ROS and ABA signalling pathways at the transcriptional level. Altogether, these results shed new light on the crosstalk between ROS and plant hormones in seed germination.