As part of the lampenflora that inhabit limestone caves, microalgae play an important role in cave ecosystems but are understudied in tropical ecoregions. In the present study, the dominant eukaryotic and prokaryotic microalgae identified in lampenflora samples collected from Gua Tempurung, a cave in Malaysia, and growth stage-related microalgal attributes were determined. Stichococcus bacillaris, Synechococcus sp., and Trentepohlia aurea were selected and cultured in Bold's Basal Medium (S. bacillaris and T. aurea) or BG-11 medium (Synechococcus sp.) under laboratory conditions. The highest specific growth rate (0.72 ± 0.21 day-1) and dry weight (0.11 ± 0.04 mg L-1) were recorded in S. bacillaris in the early stationary phase. Trentepohlia aurea and Synechococcus sp. had the highest ash-free dry weight and total ash percentage (11.18 ± 4.64 mg L-1 and 8.55% ± 6.73%, respectively) in the early stationary phase. Stichococcus bacillaris had the highest moisture content (84.26% ± 0.64%) in the exponential phase. Chlorophylls a and b were highest in the early stationary phase in T. aurea (0.706 ± 0.40 mg L-1 and 1.094 ± 0.589 mg L-1, respectively). Carotenoid levels were highest in Synechococcus sp. in the early stationary stage (0.07 ± 0.02 mg L-1). Lipids were the major biochemical compound identified at the highest levels in Synechococcus sp. (67.87% ± 7.75%) in the early stationary phase, followed by protein recorded at the highest levels in T. aurea (57.99% ± 4.99%) in the early stationary phase. Carbohydrates were the compound identified least often with the highest recorded levels found in T. aurea (9.94% ± 0.49%) in the late stationary phase. Biomass, pigments, and biochemical accumulation varied at different growth stages in the studied microalgae, and this variation was species-specific. The present study provides a benchmark for the growth phases of aerophytic cave microalgae, which will be useful for determining their optimum harvest time and obtaining biochemical compounds of interest.
Oil palm is grown in tropical soils with low bioavailability of Pi. A cDNA clone specifically expressed under phosphate-starvation condition in oil palm roots was identified as a high-affinity phosphate transporter (EgPHT1). The deduced amino acid sequence has 6 transmembrane domains each at the N- and C-termini separated by a hydrophilic linker. Comparison of promoter motifs within 1500 bp upstream of ATG of 10 promoters from high- and low-affinity phosphate transporter from both dicots and monocots including EgPHT1 was performed. The EgPHT1 promoter was fused to β-glucuronidase (GUS) reporter gene and its activity was analysed by histochemical and fluorometric GUS assays in transiently transformed oil palm tissues and T3 homozygous transgenic Arabidopsis plants. In response to Pi-starvation, no GUS activity was detected in oil palm leaves, but a strong inducible activity was observed in the roots (1.4 times higher than the CaMV35S promoter). GUS was specifically expressed in transgenic Arabidopsis roots under Pi deficiency and starvation of the other macronutrients (N and K) did not induce GUS activity. Eight motifs including ABRERATCAL (abscisic-acid responsive), RHERPATEXPA7 (root hair-specific), SURECOREATSULTR11 (sulfur-deficiency response), LTRECOREATCOR15 (temperature-stress response), MYB2CONSENSUSAT and ACGTATERD1 (water-stress response) as well as two novel motifs, 3 (TAAAAAAA) and 26 (TTTTATGT) identified through pattern discovery, occur at significantly higher frequency (p
Pyricularia oryzae (P. oryzae), one of the most devastating fungal pathogens, is the cause of blast disease in rice. Infection with a blast fungus induces biological responses in the host plant that lead to its survival through the termination or suppression of pathogen growth, and metabolite compounds play vital roles in plant interactions with a wide variety of other organisms. Numerous studies have indicated that rice has a multi-layered plant immune system that includes pre-developed (e.g., cell wall and phytoanticipins), constitutive and inducible (phytoalexins) defence barriers against stresses. Significant progress towards understanding the basis of the molecular mechanisms underlying the defence responses of rice to P. oryzae has been achieved. Nonetheless, even though the important metabolites in the responses of rice to pathogens have been identified, their exact mechanisms and their contributions to plant immunity against blast fungi have not been elucidated. The purpose of this review is to summarize and discuss recent advances towards the understanding of the integrated metabolite variations in rice after P. oryzae invasion.
Dehydration-responsive element binding (DREB) transcription factor plays an important role in controlling the expression of abiotic stress responsive genes. An intronless oil palm EgDREB1 was isolated and confirmed to be a nuclear localized protein. Electrophoretic mobility shift and yeast one-hybrid assays validated its ability to interact with DRE/CRT motif. Its close evolutionary relation to the dicot NtDREB2 suggests a universal regulatory role. In order to determine its involvement in abiotic stress response, functional characterization was performed in oil palm seedlings subjected to different levels of drought severity and in EgDREB1 transgenic tomato seedlings treated by abiotic stresses. Its expression in roots and leaves was compared with several antioxidant genes using quantitative real-time PCR. Early accumulation of EgDREB1 in oil palm roots under mild drought suggests possible involvement in the initiation of signaling communication from root to shoot. Ectopic expression of EgDREB1 in T1 transgenic tomato seedlings enhanced expression of DRE/CRT and non-DRE/CRT containing genes, including tomato peroxidase (LePOD), ascorbate peroxidase (LeAPX), catalase (LeCAT), superoxide dismutase (LeSOD), glutathione reductase (LeGR), glutathione peroxidase (LeGP), heat shock protein 70 (LeHSP70), late embryogenesis abundant (LeLEA), metallothionine type 2 (LeMET2), delta 1-pyrroline-5- carboxylate synthetase (LePCS), ABA-aldehyde oxidase (LeAAO) and 9-cis- Epoxycarotenoid dioxygenase (LeECD) under PEG treatment and cold stress (4 °C). Altogether, these findings suggest that EgDREB1 is a functional regulator in enhancing tolerance to drought and cold stress.
Agronomic crops can benefit from the application of nanoscale materials in order to control phytopathogens and improve plant growth. Bipolaris sorokiniana, a soil- and seed-borne fungus, causes severe yield losses in wheat. In order to determine the physio-chemical changes in wheat under biotic stress of B. sorokiniana, the current study aimed to synthesis silver nanoparticles (AgNPs) using Allium sativum bulb extract. Herein, we applied the silver nanoparticles (AgNPs) as a foliar spray on two wheat varieties (Pakistan-2013, and NARC-2011) at the concentrations of 10, 20, 30, and 40 mg/L to suppress B. sorokiniana. Among all the applied concentrations of AgNPs, the 40 mg/L concentration demonstrated the most effective outcome in reduction of the intensity of spot blotch and improved the morphological, physiological, biochemical parameters, as well as antioxidant activity in wheat plant. Foliar application of AgNPs at 40 mg/L Pakistan-2013 and NARC-2011 wheat varieties significantly increased chlorophyll a 84.8% and 53.4%, chlorophyll b 28.9% and 84.3%, total chlorophyll content 294.3% and 241.2%, membrane stability index 7.5% and 6.1%, relative water contents 25.4% and 10.5%, proline content 320.5% and 609.9%, and soluble sugar content 120% and 259.4%, respectively, compared to control and diseased plant. This is the first study provides important insights into the role of phyto-mediated AgNPs in increasing resistant of wheat infected with B. sorokiniana. These findings offers valuable new insights that may be useful for reducing disease incidence in wheat fields.
The identification of plant metabolites is very important for the understanding of plant physiology including plant growth, development and defense mechanism, particularly for herbal medicinal plants. The metabolite profile could possibly be used for future drug discovery since the pharmacological activities of the indigenous herbs have been proven for centuries. An untargeted mass spectrometric approach was used to identify metabolites from the leaves and stems of Impatiens balsamina using LC-DAD-MS/MS. The putative compounds are mostly from the groups of phenolic, organic and amino acids which are essential for plant growth and as intermediates for other compounds. Alanine appeared to be the main amino acid in the plant because many alanine derived metabolites were detected. There are also several secondary metabolites from the groups of benzopyrones, benzofuranones, naphthoquinones, alkaloids and flavonoids. The widely reported bioactive components such as kaempferol, quercetin and their glycosylated, lawsone and its derivatives were detected in this study. The results also revealed that aqueous methanol could extract flavonoids better than water, and mostly, flavonoids were detected from the leaf samples. The score plots of component analysis show that there is a minor variance in the metabolite profiles of water and aqueous methanolic extracts with 21.5 and 30.5% of the total variance for the first principal component at the positive and negative ion modes, respectively.
Duckweed, a floating macrophyte, has attracted interest in various fields such as animal feedstocks and bioenergy productions. Its enriched nutritional content and rapid growth rate make it particularly promising. However, common laboratory cultures of duckweed often experience fronds layering, diminishing the efficiency of sunlight capturing due to limited surface area on conventional cultivation platforms. In this work, we aimed to address the issue of fronds layering by introducing a novel cultivation platform - a superhydrophobic coated acrylic sheet. The sheet was prepared by spray-coating a suspension of beeswax and ethanol, and its effectiveness was evaluated by comparing the growth performance of giant duckweed, Spirodela polyrhiza, on this platform with that on a modified version. The superhydrophobic coated acrylic sheet (SHPA) and its variant with a metal mesh added (SHPAM) were employed as growing platforms, with a glass jar serving as the control. The plantlets were grown for 7 days with similar growth conditions under low light stress (25 μmol/m2/s). SHPAM demonstrated superior growth performance, achieving a mass gain of 102.12 ± 17.18 %, surpassing both SHPA (89.67 ± 14.97 %) and the control (39.26 ± 8.94 %). For biochemical compositions, SHPAM outperformed in chlorophyll content, protein content and lipid content. The values obtained were 1.021 ± 0.076 mg/g FW, 14.59 ± 0.58 % DW and 6.21 ± 0.75 % DW respectively. Therefore, this work proved that incorporation of superhydrophobic coatings on a novel cultivation platform significantly enhanced the biomass production of S. polyrhiza. Simultaneously, the biochemical compositions of the duckweeds were well-maintained, showcasing the potential of this approach for optimized duckweed cultivation.
Strigolactones (SLs) have been implicated in various developmental processes of the plant, including the response against several abiotic stresses. It is well known as a class of endogenous phytohormones that regulates shoot branching, secondary growth and root morphology. This hormone facilitates plants in responding to nitrogen and phosphorus starvation by shaping the above and below ground structural design. SLs actively participate within regulatory networks of plant stress adaptation that are governed by phytohormones. Heavy metals (HMs) in soil are considered a serious environmental problem that causes various harmful effects on plants. SLs along with other plant hormones imply the role in plant architecture is far from being fully understood. Strategy to remove/remediation of HMs from the soil with the help of SLs has not been defined yet. Therefore, the present review aims to comprehensively provide an overview of SLs role in fine-tuning plant architectures, relation with other plant hormones under abiotic stress, and remediation of HMs contaminated soil using SLs.
Conformational analysis of champedak galactose-binding (CGB) lectin under different urea concentrations was studied in phosphate-buffered saline (pH 7.2) using far-ultraviolet circular dichroism (far-UV CD), tryptophan (Trp) fluorescence and ANS fluorescence. In all cases, CGB lectin displayed a two-step, three-state transition. The first transition (from the native state to the intermediate state) started at ∼2.0 M urea and ended at ∼4.5 M urea, while the second transition (from the intermediate state to the completely denatured state) was characterized by the start- and end-points at ∼5.75 M and ∼7.5 M urea, respectively, when analyzed by the emission maximum of Trp fluorescence. A marked increase in the Trp fluorescence, ANS fluorescence and -CD values at 218 nm (-CD218 nm) represented the first transition, whereas a decrease in these parameters defined the second transition. On the other hand, emission maximum of the Trp fluorescence showed a continuous increase throughout the urea concentration range. Transformation of tetramer into monomer represented the first transition, whereas the second transition reflected the unfolding of monomer. Far-UV CD, Trp fluorescence and ANS fluorescence spectra were used to characterize the native, the intermediate and the completely denatured states of CGB lectin, obtained at 0.0 M, 5.0 M and 9.0 M urea, respectively. The intermediate state was characterized by the presence of higher secondary structures, increased ANS binding as well as increased Trp fluorescence intensity. A gradual decrease in the hemagglutination activity of CGB lectin was observed with increasing urea concentrations, showing complete loss at 4.0 M urea.
The present study represents the first report of the effect of hydrogen peroxide (H(2)O(2)) on the growth, development and quality of the wax apple fruit, a widely cultivated fruit tree in South East Asia. The wax apple trees were spray treated with 0, 5, 20 and 50 mM H(2)O(2) under field conditions. Photosynthetic rates, stomatal conductance, transpiration, chlorophyll and dry matter content of the leaves and total soluble solids and total sugar content of the fruits of wax apple (Syzygium samarangense, var. jambu madu) were significantly increased after treatment with 5 mM H(2)O(2). The application of 20 mM H(2)O(2) significantly reduced bud drop and enhanced fruit growth, resulting in larger fruit size, increased fruit set, fruit number, fruit biomass and yield compared to the control. In addition, the endogenous level of H(2)O(2) in wax apple leaves increased significantly with H(2)O(2) treatments. With regard to fruit quality, 20 mM H(2)O(2) treatment increased the K(+), anthocyanin and carotene contents of the fruits by 65%, 67%, and 41%, respectively. In addition, higher flavonoid, phenol and soluble protein content, sucrose phosphate synthase (SPS), phenylalanine ammonia lyase (PAL) and antioxidant activities were recorded in the treated fruits. There was a positive correlation between peel colour (hue) and TSS, between net photosynthesis and SPS activity and between phenol and flavonoid content with antioxidant activity in H(2)O(2)-treated fruits. It is concluded that spraying with 5 and 20 mM H(2)O(2) once a week produced better fruit growth, maximising the yield and quality of wax apple fruits under field conditions.
The potential significance of the previously reported papaya (Carica papaya L.) beta-galactosidase/galactanase (beta-d-galactoside galactohydrolase; EC 3.2.1.23) isoforms, beta-gal I, II and III, as softening enzymes during ripening was evaluated for hydrolysis of pectins while still structurally attached to unripe fruit cell wall, and hemicelluloses that were already solubilized in 4 M alkali. The enzymes were capable of differentially hydrolyzing the cell wall as evidenced by increased pectin solubility, pectin depolymerization, and degradation of the alkali-soluble hemicelluloses (ASH). This enzyme catalyzed in vitro changes to the cell walls reflecting in part the changes that occur in situ during ripening. beta-Galactosidase II was most effective in hydrolyzing pectin, followed by beta-gal III and I. The reverse appeared to be true with respect to the hemicelluloses. Hemicellulose, which was already released from any architectural constraints, seemed to be hydrolyzed more extensively than the pectins. The ability of the beta-galactanases to markedly hydrolyze pectin and hemicellulose suggests that galactans provide a structural cross-linkage between the cell wall components. Collectively, the results support the case for a functional relevance of the papaya enzymes in softening related changes during ripening.
The natural rubber of Para rubber tree, Hevea brasiliensis, is the main crop involved in industrial rubber production due to its superior quality. The Hevea bark is commercially exploited to obtain latex, which is produced from the articulated secondary laticifer. The laticifer is well defined in the aspect of morphology; however, only some genes associated with its development have been reported. We successfully induced secondary laticifer in the jasmonic acid (JA)-treated and linolenic acid (LA)-treated Hevea bark but secondary laticifer is not observed in the ethephon (ET)-treated and untreated Hevea bark. In this study, we analysed 27,195 gene models using NimbleGen microarrays based on the Hevea draft genome. 491 filtered differentially expressed (FDE) transcripts that are common to both JA- and LA-treated bark samples but not ET-treated bark samples were identified. In the Eukaryotic Orthologous Group (KOG) analysis, 491 FDE transcripts belong to different functional categories that reflect the diverse processes and pathways involved in laticifer differentiation. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) and KOG analysis, the profile of the FDE transcripts suggest that JA- and LA-treated bark samples have a sufficient molecular basis for secondary laticifer differentiation, especially regarding secondary metabolites metabolism. FDE genes in this category are from the cytochrome (CYP) P450 family, ATP-binding cassette (ABC) transporter family, short-chain dehydrogenase/reductase (SDR) family, or cinnamyl alcohol dehydrogenase (CAD) family. The data includes many genes involved in cell division, cell wall synthesis, and cell differentiation. The most abundant transcript in FDE list was SDR65C, reflecting its importance in laticifer differentiation. Using the Basic Local Alignment Search Tool (BLAST) as part of annotation and functional prediction, several characterised as well as uncharacterized transcription factors and genes were found in the dataset. Hence, the further characterization of these genes is necessary to unveil their role in laticifer differentiation. This study provides a platform for the further characterization and identification of the key genes involved in secondary laticifer differentiation.
We have characterized an oil palm (Elaeis guineensis Jacq.) constitutive promoter that is derived from a translationally control tumor protein (TCTP) gene. The TCTP promoter was fused transcriptionally with the gusA reporter gene and transferred to monocot and dicot systems in order to study its regulatory role in a transient expression study. It was found that the 5' region of TCTP was capable of driving the gusA expression in all the oil palm tissues tested, including immature embryo, embryogenic callus, embryoid, young leaflet from mature palm, green leaf, mesocarp and stem. It could also be used in dicot systems as it was also capable of driving gusA expression in tobacco leaves. The results indicate that the TCTP promoter could be used for the production of recombinant proteins that require constitutive expression in the plant system.
Understanding the mechanism of interaction between the oil palm and its key pathogen, Ganoderma spp. is crucial as the disease caused by this fungal pathogen leads to a major loss of revenue in leading palm oil producing countries in Southeast Asia. Here in this study, we assess the morphological and biochemical changes in Ganoderma disease infected oil palm seedling roots in both resistant and susceptible progenies. Rubber woodblocks fully colonized by G. boninense were applied as a source of inoculum to artificially infect the roots of resistant and susceptible oil palm progenies. Gas chromatography-mass spectrometry was used to measure an array of plant metabolites in 100 resistant and susceptible oil palm seedling roots treated with pathogenic Ganoderma boninense fungus. Statistical effects, univariate and multivariate analyses were used to identify key-Ganoderma disease associated metabolic agitations in both resistant and susceptible oil palm root tissues. Ganoderma disease related defense shifts were characterized based on (i) increased antifungal activity in crude extracts, (ii) increased lipid levels, beta- and gamma-sitosterol particularly in the resistant progeny, (iii) detection of heterocyclic aromatic organic compounds, benzo [h] quinoline, pyridine, pyrimidine (iv) elevation in antioxidants, alpha- and beta-tocopherol (iv) degraded cortical cell wall layers, possibly resulting from fungal hydrolytic enzyme activity needed for initial penetration. The present study suggested that plant metabolites mainly lipids and heterocyclic aromatic organic metabolites could be potentially involved in early oil palm defense mechanism against G. boninense infection, which may also highlight biomarkers for disease detection, treatment, development of resistant variety and monitoring.
Plants have developed diverse physical and chemical defence mechanisms to ensure their continued growth and well-being in challenging environments. Plants also have evolved intricate molecular mechanisms to regulate their responses to biotic stress. Non-coding RNA (ncRNA) plays a crucial role in this process that affects the expression or suppression of target transcripts. While there have been numerous reviews on the role of molecules in plant biotic stress, few of them specifically focus on how plant ncRNAs enhance resistance through various mechanisms against different pathogens. In this context, we explored the role of ncRNA in exhibiting responses to biotic stress endogenously as well as cross-kingdom regulation of transcript expression. Furthermore, we address the interplay between ncRNAs, which can act as suppressors, precursors, or regulators of other ncRNAs. We also delve into the regulation of ncRNAs in response to attacks from different organisms, such as bacteria, viruses, fungi, nematodes, oomycetes, and insects. Interestingly, we observed that diverse microorganisms interact with distinct ncRNAs. This intricacy leads us to conclude that each ncRNA serves a specific function in response to individual biotic stimuli. This deeper understanding of the molecular mechanisms involving ncRNAs in response to biotic stresses enhances our knowledge and provides valuable insights for future research in the field of ncRNA, ultimately leading to improvements in plant traits.
Phosphatidylinositol 3-kinases (PI3Ks) are characterized by the presence of a C2 domain at the N-terminal end (class I, III); or at both the N-terminal and C-terminal ends (class II), sometimes including a Plextrin homology domain and/or a Ras domain. Plant PI3Ks are analogous to the class III mammalian PI3K. An N-terminal fragment (~170 aa) of the tomato PI3K regulatory domain including the C2 domain, was cloned and expressed in a bacterial system. This protein was purified to homogeneity and its physicochemical properties analyzed. The purified protein showed strong binding with monophosphorylated phosphatidylinositols, and the binding was dependent on calcium ion concentration and pH. In the overall tertiary structure of PI3K, C2 domain showed unique characteristics, having three antiparallel beta-sheets, hydrophobic regions, acidic as well as alkaline motifs, that can enable its membrane binding upon activation. To elucidate the functional significance of C2 domain, transgenic tobacco plants expressing the C2 domain of PI3K were generated. Transgenic plants showed defective pollen development and disrupted seed set. Flowers from the PI3K-C2 transgenic plants showed delayed wilting, and a decrease in ethylene production. It is likely that introduction of the PI3K-C2 segment may have interfered with the normal binding of PI3K to the membrane, delaying the onset of membrane lipid catabolism that lead to senescence.
Graphene oxide (GO) is a novel nanomaterial with distinct physical properties and significant biological applications. The use of GO in plant tissue culture offers several new properties and potential applications. This research is vital due to the growing need for innovative techniques to promote plant growth, improve plant productivity and mitigate challenges posed by environmental stressors. This study focused on the rare Cameron Highlands white strawberry plants (Fragaria x ananassa) and addressed issues such as callus production during direct shoot induction and hyperhydricity. The research aimed to investigate the effects of GO on the regeneration process and genetic stability of white strawberry plants and to use molecular markers to ensure that plants propagated in vitro are true to type. For this purpose, shoot tip explants were used and different concentrations of GO (0, 2.5, 5.0, 7.5, 10 mg/L) were added to the Murashige and Skoog (MS) medium for six weeks. The results showed that the optimum concentration for promoting the development of white strawberry seedlings was 7.5 mg/L of GO. The study also revealed that the addition of 7.5 mg/L GO in combination with 8 μM TDZ to the MS medium facilitated the induction of multiple shoots. Moreover, the clonal fidelity of the in vitro plants treated with GO showed a genetic similarity of over 97%. These results confirm that lower GO concentrations improve plant development and stability. Consequently, this nanomaterial has a positive effect on the growth of strawberry plants and is therefore well suited for strawberry tissue culture.
The present study was conducted to investigate the effects of Trichoderma harzianum and Bacillus thuringiensis alone or with gradual levels of NPK on photosynthesis, growth, fruit quality, aroma improvement and reduced radionuclides of key lime fruits. The lemon seedlings were treated with (T0) without fertilizers as control, (T1) 100g of NPK at 100%, (T2) 5 g of Trichoderma. harzianum at 50% + 50 g of NPK at 50%, (T3) 5 g of Bacillus thuringiensis at 50% + 50 g of NPK at 50 %, (T4) 7.5 g of Trichoderma harzianum at 75% + 25 g of NPK at 25 %, (T5) 7.5 g of Bacillus thuringiensis at 75% + 25 g of NPK at 25 %, (T6) 10 g of Trichoderma harzianum at 100 % and (T7)10 g of Bacillus thuringiensis at 100 %. The results showed that T2 increased net photosynthetic rate, stomatal conductance, transpiration rate, internal CO2 concentration, fresh and dry root biomass by 209%, 74%, 56%, 376%, 69.4% and 71.6%, while, T5 increased root volume, root length, and root tip number by 27.1%, 167%, and 67%, respectively over the control trees. The microbial treatments developed cortex, vascular cylinder and tracheal elements of the root. Fruit number, length, diameter, weight, pulp thickness, pulp/peel ratio, juice, total soluble solids (TSS), pigment contents and antioxidant activity increased significantly in the T2 treatment. Vitamin C, total phenols, total flavonoids, and total sugar content increased by 1.59-, 1.66-, 1.44- and 2.07- fold in T5 treated fruits compared to the control. The two microbes increased volatile compounds and decreased radionucleotides in the fruit, moreover, 27 identified and 2 (two) unmatched volatile compounds were identified by GCMS analysis. It is concluded that T. harzianum and B. thuringiensis with 25-50 g NPK treatments improved photosynthesis, root structure, fruit growth, fruit quality, aroma and lessened radionuclides in key lime fruits.
The juvenile hormones (JH) in plants are suggested to act as a form of plant defensive strategy especially against insect herbivory. The oxidation of farnesol to farnesoic acid is a key step in the juvenile hormone biosynthesis pathway. We herein present the purification and characterisation of the recombinant Theobroma cacao farnesol dehydrogenase enzyme that catalyses oxidation of farnesol to farnesal. The recombinant enzyme was purified to apparent homogeneity by affinity chromatography. The purified enzyme was characterised in terms of its deduced amino acid sequences, phylogeny, substrate specificity, kinetic parameters, structural modeling, and docking simulation. The phylogenetic analysis indicated that the T. cacao farnesol dehydrogenase (TcFolDH) showed a close relationship with A. thaliana farnesol dehydrogenase gene. The TcFolDH monomer had a large N-terminal domain which adopted a typical Rossmann-fold, harboring the GxxGxG motif (NADP(H)-binding domain) and a small C-terminal domain. The enzyme was a homotrimer comprised of subunits with molecular masses of 36 kDa. The TcFolDH was highly specific to NADP+ as coenzyme. The substrate specificity studies showed trans, trans-farnesol was the most preferred substrate for the TcFolDH, suggesting that the purified enzyme was a NADP+-dependent farnesol dehydrogenase. The docking of trans, trans-farnesol and NADP+ into the active site of the enzyme showed the important residues, and their interactions involved in the substrate and coenzyme binding of TcFolDH. Considering the extensive involvement of JH in both insects and plants, an in-depth knowledge on the recombinant production of intermediate enzymes of the JH biosynthesis pathway could help provide a potential method for insect control.
Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp280, Leu294 and Ala303). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily.