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Simple Summary: Vacuolar processing enzyme (VPE) is a cysteine protease responsible for vacuolar proteins' maturation and regulation of programmed cell death in plants. Among four isoforms of Arabidopsis thaliana VPEs, only the functions of βVPE, γVPE, and δVPE were determined. Hence, to investigate the possible function of αVPE, promoter analysis, co-expression network, gene expression profiling, and loss of function studies were performed. Repetitive drought-related cis-elements such as ABRE, MBS, MYC, and MYB were successfully identified with the aid of PlantCARE and PLACE databases. Similarly, the co-expression network also revealed that genes interacting with αVPE were involved in drought-regulation-related function. In addition, A. thaliana under drought treatment recorded an upregulation of αVPE expression (2.7-fold). Loss of function study through αvpe knockout mutants showed that αvpe mutants remained viable with 22% higher water retention as compared with wild-type after drought treatment. Biochemical analyses recorded a 47% reduction in proline activity, 70% decrease in sucrose content, and 39% lower MDA content, but 50% increased photosynthetic pigments in αvpe mutants. Altogether, our study provided important proof and a foundation for understanding the involvement of αVPE in modulating drought tolerance in A. thaliana. 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 βVPE, 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. [ABSTRACT FROM AUTHOR]

Vandachostylis 'Sri-Siam' is a fragrant orchid, suitable as a potted plant for the floriculture and perfumery industries due to its sweet citrus-like fragrance. Fragrance from the flowers was captured using SPME and subjected to GC–MS analysis. A transcriptome library was constructed from the tepals for sequencing using the Illumina MiSeq Desktop Sequencer and for de novo assembly using the CLC Genomic Workbench version 6.0. All de novo assembly sequences were subjected to bioinformatics analyses including BLASTx analysis, functional annotations and mapping of the annotated genes/gene products to the KEGG database using the Blast2go Pro software. GC–MS analysis reveals the fragrance are dominated by 68.73% monoterpenes, 11.26% esters, 4.20% sesquiterpenes and traces of alcohols (1.20%), dissimilar to both its parents. Analysis of the de novo assembly sequences generated 45,585 contigs which identified 14 fragrance-related transcripts. Information on the fragrance constituents and fragrance-related transcripts is deemed important as a reference for current and future work of using this hybrid in the breeding process of commercially valuable fragrant orchids. [ABSTRACT FROM AUTHOR]

Vanda 'Tan Chay Yan', a beautiful golden-orange orchid with mild fragrance, is valuable in the floral industry. In this study, the volatile compounds from the flowers were profiled and a transcriptome library was created to search for fragrance-related transcripts. Volatiles from the tepals were captured using a SPME device with PDMS-coated fibre for GC-MS analysis. A transcriptome library was constructed and sequenced using the Illumina MiSeq Desktop Sequencer, de novo assembled using CLC Genomic Workbench version 6.0. BLASTx analysis, functional annotations and gene mapping of the annotated genes to the KEGG database were carried out using the Blast2go Pro software. Twelve compounds were identified in the volatiles including monoterpenes (93.19%), sesquiterpenes (4.32%) and traces of other compounds (2.49%) whereby cis-β-ocimene topped the list as the major constituent (88.41%). De novo assembly of the transcriptome generated a total of 34,027 sequences from which 20 fragrance-related transcripts in the terpenoid and benzenoid/phenylpropanoid biosynthetic pathways were identified. Combined information garnered from the volatiles and fragrance-related transcripts highlights the basic information on the fragrance constituents and biosynthetic pathways of this orchid which are useful for orchid breeders in selection of parents for new hybrids with fragrance as their value-added characteristic for the floriculture industry. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) 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. Foc TR4 infection induced the majority of MaVPE expressions in the susceptible cultivar "Berangan" as compared to the resistant cultivar "Jari Buaya." Consistently, upon Foc TR4 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 Foc TR4 infection. Additionally, the Arabidopsis VPE -null mutant exhibited higher tolerance to Foc TR4 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 Foc TR4 colonization in the host. [ABSTRACT FROM AUTHOR]

Plectranthus amboinicus (Lour.) Spreng, also known locally as "Bangun-bangun", is an aromatic medicinal herb known for its therapeutic and nutritional properties attributed to its terpenoid-rich phytochemicals. Information to assist in initiating appropriate harvesting time to maximize the yield of targeted chemicals in harvested plant tissues remains an issue that is seldom highlighted. This study reports on the essential oil distribution in P. amboinicus leaves, and total phenolic and flavonoid contents, in addition to GC-MS analysis of hexane extracts of the leaf samples collected at various times throughout the day. The influence of environmental factors on γ-terpinene, p-cymene, carvacrol, and thymoquinone are also discussed. Oil Red O staining showed the highest oil deposition at 2 p.m., which was consistent with the phenolic and flavonoid contents of this plant. GC-MS analysis of the leaf extract showed carvacrol (47.00–60.00%), γ-terpinene (8.00–10.00%), caryophyllene (~6.00%), p-cymene (4.90–6.50%), trans-α-bergamotene (4.70–5.00%), and thymoquinone (3.30–5.60%) were the major components of this plant. Interestingly, thymoquinone, a phytochemical associated with Nigella sativa, was also detected in this hexane-extracted sample with maximum accumulation during midday and a decrease at night, which could be due to the lower temperature and dimmer light conditions. The chemical polymorphism in the oil content indicated that environmental factors such as light exposure and temperature should be considered during harvesting to ensure consistent quality of the phytochemicals extracted from the plant materials. This study indicates that oversight in selecting plant materials might compromise the yield of quality phytochemicals extracted from harvested tissues. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Vanilla planifolia (V. planifolia) is a valuable orchidaceous plant, commonly grown for its pods that are used to produce the flavouring vanilla extract. Here, we evaluated the effect of calcium lignosulphonate (Ca-LIGN) and sodium lignosulphonate (Na-LIGN) on multiplication and regeneration of V. planifolia shoot tip culture. In 150 mg L−1 Ca-LIGN medium, the most number of shoots per explant (5.78 ± 0.63) was successfully obtained. Besides, Ca-LIGN also enhanced the shoot bud and primordial formation rate, as seen under scanning electron microscopy. In contrast, medium containing 150 mg L−1 Na-LIGN recorded the highest average of shoot length (4.72 ± 0.30 cm). Meanwhile, the best growth of root length (1.8 ± 0.32 cm) and root induction (96.67 ± 5.16%) were recorded on the explants treated with 150 mg L−1 Na-LIGN rooting medium. All rooted plantlets successfully acclimatized in the greenhouse (100.00% survival rate). Further biochemical analysis revealed that Ca-LIGN increased the total protein, chlorophyll, sugar, flavonoid and phenolic contents of V. planifolia. Notably, expression of both ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) genes were also elevated under the treatment of Ca-LIGN, implying a positive role in the photosynthetic process. Taken together, LIGN being an environmental friendly product could be used to enhance the growth and micropropagation of V. planifolia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Despite wide applications in industries, phenol pollution leads to many health effects, and one of the technologies used to clean up phenol pollution is phytoremediation. The aim of this research was to assess the remediation ability of Ipomoea aquatica Forssk., which is easy to handle and and has a fast growth rate. Plantlet was grown in water spiked with 0.05, 0.10, 0.20, 0.30 and 0.40 g/L phenol, followed by daily observation of the plantlets morphology and tracking of phenol concentration in the water and plantlet extracts via 4-aminoantipyrine (4-AAP) assay. Plantlet's roots in 0.10 g/L phenol (57.42 ± 1.41 mm) were significantly longer (p < 0.05) than those of the control plantlets (43.57 ± 3.87 mm) in contrast to other phenol concentrations which had stunted roots growth. I. aquatica Forssk. was able to survive with 0.30 g/L phenol despite exhibiting yellowing of leaves and increased sensitivity to scarring on the stems. The plantlets were able to completely remove the phenol from the water spiked with phenol at 0.05 g/L after 12 days of growth. However, the highest average rate of phenol removal was 0.021 g/L/day from water spiked with 0.30 g/L phenol. Phenol analysis on the plantlets' extracts revealed that I. aquatica Forssk. had degraded the absorbed phenol. This observation is of significant interest as it highlights the potential of I. aquatica Forssk. for use as a phytoremediator to clean up phenol contaminated water. [ABSTRACT FROM AUTHOR]

Isoprenoids are a large, diverse group of secondary metabolites which has recently raised a renewed research interest due to genetic engineering advances, allowing specific isoprenoids to be produced and characterized in heterologous hosts. Many researches on metabolic engineering of heterologous hosts for increased isoprenoid production are focussed on Escherichia coli and yeasts. E. coli, as most prokaryotes, use the 2-C-methyl- d-erythritol-4-phosphate (MEP) pathway for isoprenoid production. Yeasts on the other hand, use the mevalonate pathway which is commonly found in eukaryotes. However, Lactococcus lactis is an attractive alternative host for heterologous isoprenoid production. Apart from being food-grade, this Gram-positive prokaryote uses the mevalonate pathway for isoprenoid production instead of the MEP pathway. Previous studies have shown that L. lactis is able to produce sesquiterpenes through heterologous expression of plant sesquiterpene synthases. In this work, we analysed the gene expression of the lactococcal mevalonate pathway through RT- qPCR to successfully engineer L. lactis as an efficient host for isoprenoid production. We then overexpressed the mvk gene singly or co-expressed with the mvaA gene as an attempt to increase β-sesquiphellandrene production in L. lactis. It was observed that co-expression of mvk with mvaA doubled the amount of β-sesquiphellandrene produced. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The supply of agar as an important gelling and thickening agent in various industrial applications depends heavily on harvesting of natural seaweed resources and seaweed farming. To facilitate the selection of good seaweed source with higher agar yield and stronger gel strength, accurate and rapid screening method using molecular markers is necessary to replace the tedious, laborious and time-consuming conventional method which involves agar extraction and gel analysis. In this study, we characterized the expression of a number of algal transcripts and proteins from an agar producing seaweed, Gracilaria changii with the aim to identify potential markers for agar yield and gel strength. In total, 15 candidate transcripts that are directly or indirectly related to putative agar biosynthetic pathway were identified based on literature search. The transcript abundance of 4 and 11 of these candidates were found to be significantly (P < 0.05) correlated to the agar yield and gel strength of six G. changii samples, respectively. Among these marker genes, the transcript levels of GcFBPA and GcGALE have the highest linear correlation to both agar yield and gel strength. The protein abundance of GcFBPA and GcGALE was further examined on 13 G. changii samples and was found to have highly significant (P < 0.01) correlation to agar gel strength and agar yield, respectively. GcFBPA and GcGALE may have good potential to be used for molecular screening of yield traits and gel quality of G. changii at both RNA and protein levels. • The abundance of 11 transcripts correlated (P < 0.05) with agar yield and/or gel strength of G. changii. • The protein abundance of GcGALE correlated (P < 0.01) with agar yield. • The protein abundance of GcFBPA correlated (P < 0.01) with gel strength. • GcFBPA and GcGALE have potential to be markers for agar yield or gel strength. [ABSTRACT FROM AUTHOR]

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 H 2 O 2 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. • CaLS stimulates root formation in rice callus via regulation of auxin biosynthesis. • Enhancement of nutrient uptake was observed in CaLS-treated rice calli. • CaLS improves cellular homeostasis level and enhances callus proliferation rate. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]