Calcium lignosulfonate modulates physiological and biochemical responses to enhance shoot multiplication in Vanilla planifolia Andrews.

Utilisation of calcium lignosulfonate (CaLS) in Vanilla planifolia has been reported to improve shoot multiplication. However, mechanisms responsible for such observation remain unknown. Here, we elucidated the underlying mechanisms of CaLS in promoting shoot multiplication of V. planifolia via comparative proteomics, biochemical assays, and nutrient analysis. The proteome profile of CaLS-treated plants showed enhancement of several important cellular metabolisms such as photosynthesis, protein synthesis, Krebs cycle, glycolysis, gluconeogenesis, and carbohydrate synthesis. Further biochemical analysis recorded that CaLS increased Rubisco activity, hexokinase activity, isocitrate dehydrogenase activity, total carbohydrate content, glutamate synthase activity and total protein content in plant shoot, suggesting the role of CaLS in enhancing shoot growth via upregulation of cellular metabolism. Subsequent nutrient analysis showed that CaLS treatment elevated the contents of several nutrient ions especially calcium and sodium ions. In addition, our study also revealed that CaLS successfully maintained the cellular homeostasis level through the regulation of signalling molecules such as reactive oxygen species and calcium ions. These results demonstrated that the CaLS treatment can enhance shoot multiplication in V. planifolia Andrews by stimulating nutrient uptake, inducing cell metabolism, and regulating cell homeostasis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01293-w.

Promoter Cis-Element Analyses Reveal the Function of αVPE in Drought Stress Response of Arabidopsis.

Vacuolar processing enzyme (VPE) is a cysteine protease responsible for vacuolar proteins' maturation and regulation of programmed cell death (PCD). Four isoforms of Arabidopsis thaliana VPEs were identified previously, but only the functions of ßVPE, γVPE, and δVPE were determined. The specific function of a gene is linked to the cis-acting elements in the promoter region. A promoter analysis found repetitive drought-related cis-elements in αVPE, which highlight its potential involvement in drought regulation in A. thaliana. The further co-expression network portraying genes interacting with αVPE substantiated its drought-regulation-related function. Expression of αVPE was upregulated after drought treatment in A. thaliana. To confirm the role of αVPE, a loss of function study revealed that αVPE knockout mutants remained green compared with WT after drought treatment. The mutants had reduced proline activity, decreased sucrose content, and lower MDA content, but increased photosynthetic pigments, indicating that αVPE negatively regulates drought tolerance in A. thaliana. Taken together, our findings serve as important evidence of the involvement of αVPE in modulating drought tolerance in A. thaliana.

λ-Carrageenan promotes plant growth in banana via enhancement of cellular metabolism, nutrient uptake, and cellular homeostasis.

Banana (Musa acuminata) is an important fruit crop and source of income for various countries, including Malaysia. To date, current agrochemical practice has become a disputable issue due to its detrimental effect on the environment. λ-carrageenan, a natural polysaccharide extracted from edible red seaweed, has been claimed to be a potential plant growth stimulator. Hence, the present study investigates the effects of λ-carrageenan on plant growth using Musa acuminata cv. Berangan (AAA). Vegetative growth such as plant height, root length, pseudostem diameter, and fresh weight was improved significantly in λ-carrageenan-treated banana plants at an optimum concentration of 750 ppm. Enhancement of root structure was also observed in optimum λ-carrageenan treatment, facilitating nutrients uptake in banana plants. Further biochemical assays and gene expression analysis revealed that the increment in growth performance was consistent with the increase of chlorophyll content, protein content, and phenolic content, suggesting that λ-carrageenan increases photosynthesis rate, protein biosynthesis, and secondary metabolites biosynthesis which eventually stimulate growth. Besides, λ-carrageenan at optimum concentration also increased catalase and peroxidase activities, which led to a significant reduction in hydrogen peroxide and malondialdehyde, maintaining cellular homeostasis in banana plants. Altogether, λ-carrageenan at optimum concentration improves the growth of banana plants via inducing metabolic processes, enhancing nutrient uptake, and regulation of cell homeostasis. Further investigations are needed to evaluate the effectiveness of λ-carrageenan on banana plants under field conditions.

Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus.

Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10-3 µM-1 s-1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

Pluronic F-68 Improves Callus Proliferation of Recalcitrant Rice Cultivar via Enhanced Carbon and Nitrogen Metabolism and Nutrients Uptake.

Pluronic F-68 (PF-68) is a non-ionic surfactant used in plant tissue culture as a growth additive. Despite its usage as a plant growth enhancer, the mechanism underlying the growth-promoting effects of PF-68 remains largely unknown. Hence, this study was undertaken to elucidate the growth-promoting mechanism of PF-68 using recalcitrant MR 219 callus as a model. Supplementation of 0.04% PF-68 (optimum concentration) was shown to enhance callus proliferation. The treated callus recorded enhanced sugar content, protein content, and glutamate synthase activity as exemplified in the comparative proteome analysis, showing protein abundance involved in carbohydrate metabolism (alpha amylase), protein biosynthesis (ribosomal proteins), and nitrogen metabolism (glutamate synthase), which are crucial to plant growth and development. Moreover, an increase in nutrients uptake was also noted with potassium topping the list, suggesting a vital role of K in governing plant growth. In contrast, 0.10% PF-68 (high concentration) induced stress response in the callus, revealing an increment in phenylalanine ammonia lyase activity, malondialdehyde content, and peroxidase activity, which were consistent with high abundance of phenylalanine ammonia lyase, peroxidase, and peroxiredoxin proteins detected and concomitant with a reduced level of esterase activity. The data highlighted that incorporation of PF-68 at optimum concentration improved callus proliferation of recalcitrant MR 219 through enhanced carbohydrate metabolism, nitrogen metabolism, and nutrient uptake. However, growth-promoting effects of PF-68 are concentration dependent.

Sodium lignosulfonate improves shoot growth of Oryza sativa via enhancement of photosynthetic activity and reduced accumulation of reactive oxygen species.

Lignosulfonate (LS) is a by-product obtained during sulfite pulping process and is commonly used as a growth enhancer in plant growth. However, the underlying growth promoting mechanism of LS on shoot growth remains largely unknown. Hence, this study was undertaken to determine the potential application of eco-friendly ion-chelated LS complex [sodium LS (NaLS) and calcium LS (CaLS)] to enhance recalcitrant indica rice MR 219 shoot growth and to elucidate its underlying growth promoting mechanisms. In this study, the shoot apex of MR 219 rice was grown on Murashige and Skoog medium supplemented with different ion chelated LS complex (NaLS and CaLS) at 100, 200, 300 and 400 mg/L The NaLS was shown to be a better shoot growth enhancer as compared to CaLS, with optimum concentration of 300 mg/L. Subsequent comparative proteomic analysis revealed an increase of photosynthesis-related proteins [photosystem II (PSII) CP43 reaction center protein, photosystem I (PSI) iron-sulfur center, PSII CP47 reaction center protein, PSII protein D1], ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), carbohydrate metabolism-related proteins (glyceraldehyde-3-phosphate dehydrogenase 3, fructose-bisphosphate aldolase) and stress regulator proteins (peptide methionine sulfoxide reductase A4, delta-1-pyrroline-5-carboxylate synthase 1) abundance in NaLS-treated rice as compared to the control (MSO). Consistent with proteins detected, a significant increase in biochemical analyses involved in photosynthetic activities, carbohydrate metabolism and protein biosynthesis such as total chlorophyll, rubisco activity, total sugar and total protein contents were observed in NaLS-treated rice. This implies that NaLS plays a role in empowering photosynthesis activities that led to plant growth enhancement. In addition, the increased in abundance of stress regulator proteins were consistent with low levels of peroxidase activity, malondialdehyde content and phenylalanine ammonia lyase activity observed in NaLS-treated rice. These results suggest that NaLS plays a role in modulating cellular homeostasis to provide a conducive cellular environment for plant growth. Taken together, NaLS improved shoot growth of recalcitrant MR 219 rice by upregulation of photosynthetic activities and reduction of ROS accumulation leading to better plant growth.

An Overview of the Potential Therapeutic Applications of Essential Oils.

The emergence of antimicrobial resistance (AMR) has urged researchers to explore therapeutic alternatives, one of which includes the use of natural plant products such as essential oils (EO). In fact, EO obtained from clove, oregano, thymus, cinnamon bark, rosemary, eucalyptus, and lavender have been shown to present significant inhibitory effects on bacteria, fungi, and viruses; many studies have been done to measure EO efficacy against microorganisms. The strategy of combinatory effects via conventional and non-conventional methods revealed that the combined effects of EO-EO or EO-antibiotic exhibit enhanced efficacy. This paper aims to review the antimicrobial effects of EO, modes of EO action (membrane disruption, efflux inhibition, increase membrane permeability, and decrease in intracellular ATP), and their compounds' potential as effective agents against bacteria, fungi, and viruses. It is hoped that the integration of EO applications in this work can be used to consider EO for future clinical applications.

Calcium lignosulfonate improves proliferation of recalcitrant indica rice callus via modulation of auxin biosynthesis and enhancement of nutrient absorption.

Lignosulfonate (LS) is a commonly used to promote plant growth. However, the underlying growth promoting responses of LS in plant remain unknown. Therefore, this study was undertaken to elucidate the underlying growth promoting mechanisms of LS, specifically calcium lignosulfonate (CaLS). Addition of 100 mg/L CaLS in phytohormone-free media enhanced recalcitrant indica rice cv. MR219 callus proliferation rate and adventitious root formation. Both, auxin related genes (OsNIT1, OsTAA1 and OsYUC1) and tryptophan biosynthesis proteins were upregulated in CaLS-treated calli which corroborated with increased of endogenous auxin content. Moreover, increment of OsWOX11 gene on CaLS-treated calli implying that the raised of endogenous auxin was utilized as a cue to enhance adventitious root development. Besides, CaLS-treated calli showed higher nutrient ions content with major increment in calcium and potassium ions. Consistently, increased of potassium protein kinases genes (OsAKT1, OsHAK5, OsCBL, OsCIPK23 and OsCamk1) were also recorded. In CaLS treated calli, the significant increase of calcium ion was observed starting from week one while potassium ion only recorded significant increase on week two onwards, suggesting that increment of potassium ion might be dependent on the calcium ion content in the plant cell. Additionally, reduced callus blackening was also coherent with downregulation of ROS scavenging protein and reduced H2O2 content in CaLS-treated calli suggesting the role of CaLS in mediating cellular homeostasis via prevention of oxidative burst in the cell. Taken together, CaLS successfully improved MR219 callus proliferation and root formation by increasing endogenous auxin synthesis, enhancing nutrients uptake and regulating cellular homeostasis.

Vacuolar Processing Enzymes Modulating Susceptibility Response to Fusarium oxysporum f. sp. cubense Tropical Race 4 Infections in Banana.

Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4) is a destructive necrotrophic fungal pathogen afflicting global banana production. Infection process involves the activation of programmed cell death (PCD). In this study, seven Musa acuminata vacuolar processing enzyme (MaVPE1-MaVPE7) genes associated with PCD were successfully identified. Phylogenetic analysis and tissue-specific expression categorized these MaVPEs into the seed and vegetative types. FocTR4 infection induced the majority of MaVPE expressions in the susceptible cultivar "Berangan" as compared to the resistant cultivar "Jari Buaya." Consistently, upon FocTR4 infection, high caspase-1 activity was detected in the susceptible cultivar, while low level of caspase-1 activity was recorded in the resistant cultivar. Furthermore, inhibition of MaVPE activities via caspase-1 inhibitor in the susceptible cultivar reduced tonoplast rupture, decreased lesion formation, and enhanced stress tolerance against FocTR4 infection. Additionally, the Arabidopsis VPE-null mutant exhibited higher tolerance to FocTR4 infection, indicated by reduced sporulation rate, low levels of H2O2 content, and high levels of cell viability. Comparative proteomic profiling analysis revealed increase in the abundance of cysteine proteinase in the inoculated susceptible cultivar, as opposed to cysteine proteinase inhibitors in the resistant cultivar. In conclusion, the increase in vacuolar processing enzyme (VPE)-mediated PCD played a crucial role in modulating susceptibility response during compatible interaction, which facilitated FocTR4 colonization in the host.

Functional characterization of a new terpene synthase from Plectranthus amboinicus.

Plectranthus amboinicus (Lour.) Spreng is an aromatic medicinal herb known for its therapeutic and nutritional properties attributed by the presence of monoterpene and sesquiterpene compounds. Up until now, research on terpenoid biosynthesis has focused on a few mint species with economic importance such as thyme and oregano, yet the terpene synthases responsible for monoterpene production in P. amboinicus have not been described. Here we report the isolation, heterologous expression and functional characterization of a terpene synthase involved in P. amboinicus terpenoid biosynthesis. A putative monoterpene synthase gene (PamTps1) from P. amboinicus was isolated with an open reading frame of 1797 bp encoding a predicted protein of 598 amino acids with molecular weight of 69.6 kDa. PamTps1 shares 60-70% amino acid sequence similarity with other known terpene synthases of Lamiaceae. The in vitro enzymatic activity of PamTps1 demonstrated the conversion of geranyl pyrophosphate and farnesyl pyrophosphate exclusively into linalool and nerolidol, respectively, and thus PamTps1 was classified as a linalool/nerolidol synthase. In vivo activity of PamTps1 in a recombinant Escherichia coli strain revealed production of linalool and nerolidol which correlated with its in vitro activity. This outcome validated the multi-substrate usage of this enzyme in producing linalool and nerolidol both in in vivo and in vitro systems. The transcript level of PamTps1 was prominent in the leaf during daytime as compared to the stem. Gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR analyses showed that maximal linalool level was released during the daytime and lower at night following a diurnal circadian pattern which correlated with the PamTps1 expression pattern. The PamTps1 cloned herein provides a molecular basis for the terpenoid biosynthesis in this local herb that could be exploited for valuable production using metabolic engineering in both microbial and plant systems.

Growth promoting effects of Pluronic F-68 on callus proliferation of recalcitrant rice cultivar.

This study was undertaken to evaluate growth-promoting effects of Pluronic F-68 (PF-68) on recalcitrant MR 219 rice callus. Our study shows that calli grown on Murashige and Skoog medium supplemented with 0.04% PF-68 significantly increased callus proliferation by 58.80% (fresh weight) and 23.98% (dry weight) while root formation from callus was enhanced by 28.57%. Enhanced callus proliferation was supported by biochemical analysis, whereby highest amount of soluble sugar (1.77 mg/mL) and protein (0.17 mg/mL) contents were recorded in calli grown on 0.04% PF-68. Furthermore, enhanced expression of sucrose synthase (2.65-folds) and NADH-dependent glutamate synthase (1.86-folds) genes in calli grown on 0.04% PF-68 also correlates with enhanced callus proliferation. In contrast, high concentration of PF-68 (0.10%) recorded highest amount of phenolic (0.74 mg/mL), flavonoid (0.08 mg/mL), and hydrogen peroxide content (0.06 mg/mL) as compared to other treatment groups indicates activation of plant defence mechanism towards stress. Similarly, high expression of 4-coumarate:CoA ligase 3 (1.28-folds), chalcone-flavonone isomerase (1.65-folds) and ascorbate peroxidase (1.61-folds) genes were observed in calli grown on 0.10% PF-68 further supports increasing stress caused by the high concentration of PF-68. Taken together, our study revealed that optimum concentration of PF-68 could improve recalcitrant rice callus proliferation via enhanced sugar metabolism and amino acid biosynthesis which are crucial towards plant growth and development. However, at high concentration, PF-68 induces stress in plant which enhance the production of secondary metabolite to maintain cellular homeostasis.

Malaysian Carica papaya L. var. Eksotika: Current Research Strategies Fronting Challenges.

Carica papaya L. or commonly known as papaya, is a major tropical crop consumed worldwide either as a vegetable or fresh fruit or processed products. In Malaysia, papaya was initially planted as a smallholder crop throughout the country. Eventually after 15 years of breeding and selection, a new variety, named C. papaya L. var. Eksotika, was released by the Malaysian Agricultural Research and Development Institute (MARDI) in 1987. This event changed the outlook of papaya planting from a smallholder crop to a plantation crop. Despite the blooming papaya business, the industry faced various disease issues that jeopardize its future. The most devastating was the papaya dieback disease, which affected approximately 800 hectares of plantation, destroyed approximately 1 million trees nationwide with total losses estimated at US$ 58 million. Even though Eksotika is a favored commercial variety with good eating and aesthetic quality fruit, its potential for more lucrative distant markets is tarnished with its short-shelf life fruits. Several strategies had been reported to address the challenges faced by Eksotika specifically against the dieback disease and the fruit's short shelf-life. This review focuses on C. papaya L. var. Eksotika particularly on the strategies to address the challenges faced in order to sustain the economic value of this crop plant, which had contributed significantly to the Malaysian economy.

Transcriptome profiling of sulfate deprivation responses in two agarophytes Gracilaria changii and Gracilaria salicornia (Rhodophyta).

Seaweeds survive in marine waters with high sulfate concentration compared to those living at freshwater habitats. The cell wall polymer of Gracilaria spp. which supplies more than 50% of the world agar is heavily sulfated. Since sulfation reduces the agar quality, it is interesting to investigate the effects of sulfate deprivation on the sulfate contents of seaweed and agar, as well as the metabolic pathways of these seaweeds. In this study, two agarophytes G. changii and G. salicornia were treated under sulfate deprivation for 5 days. The sulfate contents in the seaweed/agar were generally lower in sulfate-deprivated samples compared to those in the controls, but the differences were only statistically significant for seaweed sample of G. changii and agar sample of G. salicornia. RNA sequencing (RNA-Seq) of sulfate-deprivated and untreated seaweed samples revealed 1,292 and 3,439 differentially expressed genes (DEGs; ≥1.5-fold) in sulfate-deprivated G. changii and G. salicornia, respectively, compared to their respective controls. Among the annotated DEGs were genes involved in putative agar biosynthesis, sulfur metabolism, metabolism of sulfur-containing amino acids, carbon metabolism and oxidative stress. These findings shed light on the sulfate deprivation responses in agarophytes and help to identify candidate genes involved in agar biosynthesis.

Biosynthesis of agar in red seaweeds: A review.

Agar is a jelly-like biopolymer synthesized by many red seaweeds as their major cell wall component. Due to its excellent rheological properties, it has been exploited commercially for applications in food, cosmetic, pharmaceutical, biomedical and biotechnology industries. Despite its multiple uses, the biosynthesis of this phycocolloid is not fully understood. The current knowledge on agar biosynthesis is inferred from plant biochemistry and putative pathways for ulvan and alginate biosynthesis in green and brown seaweeds, respectively. In this review, the gaps in our current knowledge on agar biosynthetic pathway are discussed, focusing on the biosynthesis of agar precursors, elongation of agar polysaccharide chain and side chain modification. The development of molecular markers for the screening of desired seaweeds for industrial exploitation is also discussed.

Effects of Flower and Fruit Extracts of Melastoma malabathricum Linn. on Growth of Pathogenic Bacteria: Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium.

Melastoma malabathricum Linn. is a shrub that comes with beautiful pink or purple flowers and has berries-like fruits rich in anthocyanins. This study was carried out with the aim to evaluate the inhibitory activities of different concentrations of the M. malabathricum Linn. flower and fruit crude extracts against Listeria monocytogenes IMR L55, Staphylococcus aureus IMR S244, Escherichia coli IMR E30, and Salmonella typhimurium IMR S100 using the disc diffusion method. The lowest concentrations of the extracts producing inhibition zones against the test microorganisms were used to determine their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). In addition, the growth of Listeria monocytogenes IMR L55 and Staphylococcus aureus IMR S244 grown in medium supplemented with the respective extracts at different temperatures (4°C, 25°C, and 37°C) and pHs (4, 6, 7, and 8) was determined.