Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and β-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to β-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.
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.
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.
Abiotic stress reduces plant growth and crop productivity. However, the mechanism underlying posttranscriptional regulations of stress response remains elusive. Herein, we report the posttranscriptional mechanism of nucleocytoplasmic RNA transport of stress-responsive transcripts mediated by EgRBP42, a heterogeneous nuclear ribonucleoprotein-like RNA-binding protein from oil palm, which could be necessary for rapid protein translation to confer abiotic stress tolerance in plants. Transgenic Arabidopsis overexpressing EgRBP42 showed early flowering through alteration of gene expression of flowering regulators and exhibited tolerance towards heat, cold, drought, flood, and salinity stresses with enhanced poststress recovery response by increasing the expression of its target stress-responsive genes. EgRBP42 harbours nucleocytoplasmic shuttling activity mediated by the nuclear localization signal and the M9-like domain of EgRBP42 and interacts directly with regulators in the nucleus, membrane, and the cytoplasm. EgRBP42 regulates the nucleocytoplasmic RNA transport of target stress-responsive transcripts through direct binding to their AG-rich motifs. Additionally, EgRBP42 transcript and protein induction by environmental stimuli are regulated at the transcriptional and posttranscriptional levels. Taken together, the posttranscriptional regulation of RNA transport mediated by EgRBP42 may change the stress-responsive protein profiles under abiotic stress conditions leading to a better adaptation of plants to environmental changes.
MAIN CONCLUSION: Rice sheath blight research should prioritise optimising biological control approaches, identification of resistance gene mechanisms and application in genetic improvement and smart farming for early disease detection. Rice sheath blight, caused by Rhizoctonia solani AG1-1A, is one of the most devasting diseases of the crop. To move forward with effective crop protection against sheath blight, it is important to review the published information related to pathogenicity and disease management and to determine areas of research that require deeper study. While progress has been made in the identification of pathogenesis-related genes both in rice and in the pathogen, the mechanisms remain unclear. Research related to disease management practices has addressed the use of agronomic practices, chemical control, biological control and genetic improvement: Optimising nitrogen fertiliser use in conjunction with plant spacing can reduce spread of infection while smart agriculture technologies such as crop monitoring with Unmanned Aerial Systems assist in early detection and management of sheath blight disease. Replacing older fungicides with natural fungicides and use of biological agents can provide effective sheath blight control, also minimising environmental impact. Genetic approaches that show promise for the control of sheath blight include treatment with exogenous dsRNA to silence pathogen gene expression, genome editing to develop rice lines with lower susceptibility to sheath blight and development of transgenic rice lines overexpressing or silencing pathogenesis related genes. The main challenges that were identified for effective crop protection against sheath blight are the adaptive flexibility of the pathogen, lack of resistant rice varieties, abscence of single resistance genes for use in breeding and low access of farmers to awareness programmes for optimal management practices.
MicroRNAs (miRNAs) play critical regulatory roles by acting as sequence specific guide during secondary wall formation in woody and non-woody species. Although thousands of plant miRNAs have been sequenced, there is no comprehensive view of miRNA mediated gene regulatory network to provide profound biological insights into the regulation of xylem development. Herein, we report the involvement of six highly conserved amg-miRNA families (amg-miR166, amg-miR172, amg-miR168, amg-miR159, amg-miR394, and amg-miR156) as the potential regulatory sequences of secondary cell wall biosynthesis. Within this highly conserved amg-miRNA family, only amg-miR166 exhibited strong differences in expression between phloem and xylem tissue. The functional characterization of amg-miR166 targets in various tissues revealed three groups of HD-ZIP III: ATHB8, ATHB15, and REVOLUTA which play pivotal roles in xylem development. Although these three groups vary in their functions, -psRNA target analysis indicated that miRNA target sequences of the nine different members of HD-ZIP III are always conserved. We found that precursor structures of amg-miR166 undergo exhaustive sequence variation even within members of the same family. Gene expression analysis showed three key lignin pathway genes: C4H, CAD, and CCoAOMT were upregulated in compression wood where a cascade of miRNAs was downregulated. This study offers a comprehensive analysis on the involvement of highly conserved miRNAs implicated in the secondary wall formation of woody plants.
Association analysis was conducted in a core collection of 94 genotypes of Solanum pimpinellifolium to identify variations linked to salt tolerance traits (physiological and yield traits under salt stress) in four candidate genes viz., DREB1A, VP1.1, NHX1, and TIP. The candidate gene analysis covered a concatenated length of 4594 bp per individual and identified five SNP/Indels in DREB1A and VP1.1 genes explaining 17.0% to 25.8% phenotypic variation for various salt tolerance traits. Out of these five alleles, one at 297 bp in DREB1A had in-frame deletion of 6 bp (CTGCAT) or 12 bp (CTGCATCTGCAT), resulting in two alleles, viz., SpDREB1A_297_6 and SpDREB1A_297_12. These alleles individually or as haplotypes accounted for maximum phenotypic variance of about 25% for various salt tolerance traits. Design of markers for selection of the favorable alleles/haplotypes will hasten marker-assisted introgression of salt tolerance from S. pimpinellifolium into cultivated tomato.
The R2R3-MYB genes comprise one of the largest transcription factor gene families in plants, playing regulatory roles in plant-specific developmental processes, defense responses and metabolite accumulation. To date MYB family genes have not yet been comprehensively identified in the major staple fruit crop banana. In this study, we present a comprehensive, genome-wide analysis of the MYB genes from Musa acuminata DH-Pahang (A genome). A total of 285 R2R3-MYB genes as well as genes encoding three other classes of MYB proteins containing multiple MYB repeats were identified and characterised with respect to structure and chromosomal organisation. Organ- and development-specific expression patterns were determined from RNA-Seq data. For 280 M. acuminata MYB genes for which expression was found in at least one of the analysed samples, a variety of expression patterns were detected. The M. acuminata R2R3-MYB genes were functionally categorised, leading to the identification of seven clades containing only M. acuminata R2R3-MYBs. The encoded proteins may have specialised functions that were acquired or expanded in Musa during genome evolution. This functional classification and expression analysis of the MYB gene family in banana establishes a solid foundation for future comprehensive functional analysis of MaMYBs and can be utilized in banana improvement programmes.
The continuous and sole dependence on imidazolinone (IMI) herbicides for weedy rice control has led to the evolution of herbicide resistance in weedy rice populations across various countries growing IMI herbicide-resistant rice (IMI-rice), including Malaysia. A comprehensive study was conducted to elucidate occurrence, level, and mechanisms endowing resistance to IMI herbicides in putative resistant (R) weedy rice populations collected from three local Malaysian IMI-rice fields. Seed bioassay and whole-plant dose-response experiments were conducted using commercial IMI herbicides. Based on the resistance index (RI) quantification in both experiments, the cross-resistance pattern of R and susceptible (S) weedy rice populations and control rice varieties (IMI-rice variety MR220CL2 and non-IMI-rice variety MR219) to imazapic and imazapyr was determined. A molecular investigation was carried out by comparing the acetohydroxyacid synthase (AHAS) gene sequences of the R and S populations and the MR220CL2 and MR219 varieties. The AHAS gene sequences of R weedy rice were identical to those of MR220CL2, exhibiting a Ser-653-Asn substitution, which was absent in MR219 and S plants. In vitro assays were conducted using analytical grade IMI herbicides of imazapic (99.3%) and imazapyr (99.6%) at seven different concentrations. The results demonstrated that the AHAS enzyme extracted from the R populations and MR220CL2 was less sensitive to IMI herbicides than that from S and MR219, further supporting that IMI herbicide resistance was conferred by target-site mutation. In conclusion, IMI resistance in the selected populations of Malaysian weedy rice could be attributed to a Ser-653-Asn mutation that reduced the sensitivity of the target site to IMI herbicides. To our knowledge, this study is the first to show the resistance mechanism in weedy rice from Malaysian rice fields.
Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards farnesal. Thus, it was suggested that this novel enzyme may be functioning specifically to oxidize farnesal in the later steps of JH III pathway. This report provides a basic understanding for recombinant production of this particular enzyme. Other strategies such as adding His-tag to the protein makes easy the purification of the protein which is completely different to the native protein. Complete sequence, structure and functional analysis of the enzyme will be important for developing insect-resistant crop plants by deployment of transgenic plant.
To investigate limiters of photosynthate assimilation in the carbon-source limited crop, oil palm (Elaeis guineensis Jacq.), we measured differential metabolite, gene expression and the gas exchange in leaves in an open field for palms with distinct mesocarp oil content. We observed higher concentrations of glucose 1-phosphate, glucose 6-phosphate, sucrose 6-phosphate, and sucrose in high-oil content palms with the greatest difference being at 11:00 (p-value ≤0.05) immediately after the period of low morning light intensity. Three important photosynthetic genes were identified using differentially expressed gene analysis (DEGs) and were found to be significantly enriched through Gene Ontology (GO) and pathway enrichment: chlorophyll a-b binding protein (CAB-13), photosystem I (PSI), and Ferredoxin-NADP reductase (FNR), particularly for sampling points at non-peak light (11:00 and 19:00), ranging from 3.3-fold (PSI) and 5.6-fold (FNR) to 10.3-fold (CAB-13). Subsequent gas exchange measurements further supported increased carbon assimilation through higher level of internal CO2 concentration (Ci), stomatal conductance (gs) and transpiration rate (E) in high-oil content palms. The selection for higher expression of key photosynthesis genes together with CO2 assimilation under low light is likely to be important for crop improvement, in particular at full maturity and under high density planting regimes where light competition exists between palms.
Plant-derived terpenoids are extensively used in perfume, food, cosmetic and pharmaceutical industries, and several attempts are being made to produce terpenes in heterologous hosts. Native hosts have evolved to accumulate large quantities of terpenes in specialized cells. However, heterologous cells lack the capacity needed to produce and store high amounts of non-native terpenes, leading to reduced growth and loss of volatile terpenes by evaporation. Here, we describe how to direct the sesquiterpene patchoulol production into cytoplasmic lipid droplets (LDs) in Physcomitrium patens (syn. Physcomitrella patens), by attaching patchoulol synthase (PTS) to proteins linked to plant LD biogenesis. Three different LD-proteins: Oleosin (PpOLE1), Lipid Droplet Associated Protein (AtLDAP1) and Seipin (PpSeipin325) were tested as anchors. Ectopic expression of PTS increased the number and size of LDs, implying an unknown mechanism between heterologous terpene production and LD biogenesis. The expression of PTS physically linked to Seipin increased the LD size and the retention of patchoulol in the cell. Overall, the expression of PTS was lower in the anchored mutants than in the control, but when normalized to the expression the production of patchoulol was higher in the seipin-linked mutants.
Water shortage is among the major abiotic stresses that restrict growth and productivity of citrus. The existing literature indicates that tetraploid rootstocks had better water-deficit tolerance than corresponding diploids. However, the associated tolerance mechanisms such as antioxidant defence and nutrient uptake are less explored. Therefore, we evaluated physiological and biochemical responses (antioxidant defence, osmotic adjustments and nutrient uptake) of diploid (2x) and tetraploid (4x) volkamer lemon (VM) rootstocks grafted with kinnow mandarin (KM) under two water-deficit regimes. The KM/4xVM (VM4) and KM/2xVM (VM2) observed decrease in photosynthetic variables, i.e., photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E), leaf greenness (SPAD), dark adopted chlorophyll fluorescence (Fv/Fm), dark adopted chlorophyll fluorescence (Fv´/Fm´), relative water contents (RWC) and leaf surface area (LSA), and increase in non-photochemical quenching (NPQ) under both water-deficit regimes. Moreover, oxidative stress indicators, i.e., malondialdehyde (MDA) and hydrogen peroxide, and activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APx), glutathione reductase (GR) were increased under both water-deficit regimes. Nonetheless, increase was noted in osmoprotectants such as proline (PRO) and glycine betaine (GB) and other biochemical compounds, including antioxidant capacity (AC), total phenolic content (TPC) and total soluble protein (TSP) in VM2 and VM4 under both water-deficit regimes. Dry biomass (DB) of both rootstocks was decreased under each water-deficit condition. Interestingly, VM4 showed higher and significant increase in antioxidant enzymes, osmoprotectants and other biochemical compounds, while VM2 exhibited higher values for oxidative stress indicators. Overall, results indicated that VM4 better tolerated water-deficit stress by maintaining photosynthetic variables associated with strong antioxidant defence machinery as compared to VM2. However, nutrient uptake was not differed among tested water-deficit conditions and rootstocks. The results conclude that VM4 can better tolerate water-deficit than VM2. Therefore, VM4 can be used as rootstock in areas of high-water deficiency for better citrus productivity.
Soil salinity exert negative impacts on agricultural production and regarded as a crucial issue in global wetland rice production (Oryza sativa L.). Indigenous salt-tolerant plant growth-promoting rhizobacteria (Bacillus sp.) could be used for improving rice productivity under salinity stress. This study screened potential salt-tolerant plant growth-promoting rhizobacteria (PGPR) collected from coastal salt-affected rice cultivation areas under laboratory and glasshouse conditions. Furthermore, the impacts of these PGPRs were tested on biochemical attributes and nutrient contents in various rice varieties under salt stress. The two most promising PGPR strains, i.e., 'UPMRB9' (Bacillus tequilensis 10b) and 'UPMRE6' (Bacillus aryabhattai B8W22) were selected for glasshouse trial. Results indicated that 'UPMRB9' improved osmoprotectant properties, i.e., proline and total soluble sugar (TSS), antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT). Moreover, 'UPMRB9' inoculated rice plants accumulated higher amount of nitrogen and calcium in tissues. Therefore, the indigenous salt-tolerant PGPR strain 'UPMRB9' could be used as a potential bio-augmentor for improving biochemical attributes and nutrient uptake in rice plants under salinity stress. This study could serve as a preliminary basis for future large-scale trials under glasshouse and field conditions.
Hevea brasiliensis remains the primary crop commercially exploited to obtain latex, which is produced from the articulated secondary laticifer. Here, we described the transcriptional events related to jasmonic acid (JA)- and linolenic acid (LA)-induced secondary laticifer differentiation (SLD) in H. brasiliensis clone RRIM 600 based on RNA-seq approach. Histochemical approach proved that JA- and LA-treated samples resulted in SLD in H. brasiliensis when compared to ethephon and untreated control. RNA-seq data resulted in 86,614 unigenes, of which 2,664 genes were differentially expressed in JA and LA-induced secondary laticifer harvested from H. brasiliensis bark samples. Among these, 450 genes were unique to JA and LA as they were not differentially expressed in ethephon-treated samples compared with the untreated samples. Most transcription factors from the JA- and LA-specific dataset were classified under MYB, APETALA2/ethylene response factor (AP2/ERF), and basic-helix-loop-helix (bHLH) gene families that were involved in tissue developmental pathways, and we proposed that Bel5-GA2 oxidase 1-KNOTTED-like homeobox complex are likely involved in JA- and LA-induced SLD in H. brasiliensis. We also discovered alternative spliced transcripts, putative novel transcripts, and cis-natural antisense transcript pairs related to SLD event. This study has advanced understanding on the transcriptional regulatory network of SLD in H. brasiliensis.
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.
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.
Basal stem rot (BSR) of oil palm is a disastrous disease caused by a white-rot fungus Ganoderma boninense Pat. Non-ribosomal peptides (NRPs) synthesized by non-ribosomal peptide synthetases (NRPSs) are a group of secondary metabolites that act as fungal virulent factors during pathogenesis in the host. In this study, we aimed to isolate NRPS gene of G. boninense strain UPMGB001 and investigate the role of this gene during G. boninense-oil palm interaction. The isolated NRPS DNA fragment of 8322 bp was used to predict the putative peptide sequence of different domains and showed similarity with G. sinense (85%) at conserved motifs of three main NRPS domains. Phylogenetic analysis of NRPS peptide sequences demonstrated that NRPS of G. boninense belongs to the type VI siderophore family. The roots of 6-month-old oil palm seedlings were artificially inoculated for studying NRPS gene expression and disease severity in the greenhouse. The correlation between high disease severity (50%) and high expression (67-fold) of G. boninense NRPS gene at 4 months after inoculation and above indicated that this gene played a significant role in the advancement of BSR disease. Overall, these findings increase our knowledge on the gene structure of NRPS in G. boninense and its involvement in BSR pathogenesis as an effector gene.
The fructose-1,6-bisphosphate aldolase catalyzed glycolysis branch that forms dihydroxyacetone phosphate and glyceraldehyde-3-phosphate was identified as a key driver of increased oil synthesis in oil palm and was validated in Saccharomyces cerevisiae. Reduction in triose phosphate isomerase (TPI) activity in a yeast knockdown mutant resulted in 19% increase in lipid content, while yeast strains overexpressing oil palm fructose-1,6-bisphosphate aldolase (EgFBA) and glycerol-3-phosphate dehydrogenase (EgG3PDH) showed increased lipid content by 16% and 21%, respectively. Genetic association analysis on oil palm SNPs of EgTPI SD_SNP_000035801 and EgGAPDH SD_SNP_000041011 showed that palms harboring homozygous GG in EgTPI and heterozygous AG in EgGAPDH exhibited higher mesocarp oil content based on dry weight. In addition, AG genotype of the SNP of EgG3PDH SD_SNP_000008411 was associated with higher mean mesocarp oil content, whereas GG genotype of the EgFBA SNP SD_SNP_000007765 was favourable. Additive effects were observed with a combination of favourable alleles in TPI and FBA in Nigerian x AVROS population (family F7) with highest allele frequency GG.GG being associated with a mean increase of 3.77% (p value = 2.3E-16) oil content over the Family 1. An analogous effect was observed in yeast, where overexpressed EgFBA in TPI - resulted in a 30% oil increment. These results provide insights into flux balances in glycolysis leading to higher yield in mesocarp oil-producing fruit.
SAG21/LEA5 is an unusual late embryogenesis abundant protein in Arabidopsis thaliana, that is primarily mitochondrially located and may be important in regulating translation in both chloroplasts and mitochondria. SAG21 expression is regulated by a plethora of abiotic and biotic stresses and plant growth regulators indicating a complex regulatory network. To identify key transcription factors regulating SAG21 expression, yeast-1-hybrid screens were used to identify transcription factors that bind the 1685 bp upstream of the SAG21 translational start site. Thirty-three transcription factors from nine different families bound to the SAG21 promoter, including members of the ERF, WRKY and NAC families. Key binding sites for both NAC and WRKY transcription factors were tested through site directed mutagenesis indicating the presence of cryptic binding sites for both these transcription factor families. Co-expression in protoplasts confirmed the activation of SAG21 by WRKY63/ABO3, and SAG21 upregulation elicited by oligogalacturonide elicitors was partially dependent on WRKY63, indicating its role in SAG21 pathogen responses. SAG21 upregulation by ethylene was abolished in the erf1 mutant, while wound-induced SAG21 expression was abolished in anac71 mutants, indicating SAG21 expression can be regulated by several distinct transcription factors depending on the stress condition.