Proteomics and Interspecies Interaction Analysis Revealed Abscisic Acid Signalling to Be the Primary Driver for Oil Palm's Response against Red Palm Weevil Infestation.

The red palm weevil (RPW; Rhynchophorus ferrugineus Olivier (Coleoptera Curculionidae)) is an invasive insect pest that is difficult to manage due to its nature of infesting the host palm trees from within. A holistic, molecular-based approach to identify proteins that correlate with RPW infestation could give useful insights into the vital processes that are prevalent to the host's infestation response and identify the potential biomarkers for an early detection technique. Here, a shotgun proteomic analysis was performed on oil palm (Elaeis guineensis; OP) under untreated (control), wounding by drilling (wounded), and artificial larval infestation (infested) conditions at three different time points to characterise the RPW infestation response at three different stages. KEGG pathway enrichment analysis revealed many overlapping pathways between the control, wounded, and infested groups. Further analysis via literature searches narrowed down biologically relevant proteins into categories, which were photosynthesis, growth, and stress response. Overall, the patterns of protein expression suggested abscisic acid (ABA) hormone signalling to be the primary driver of insect herbivory response. Interspecies molecular docking analysis between RPW ligands and OP receptor proteins provided putative interactions that result in ABA signalling activation. Seven proteins were selected as candidate biomarkers for early infestation detection based on their relevance and association with ABA signalling. The MS data are available via ProteomeXchange with identifier PXD028986. This study provided a deeper insight into the mechanism of stress response in OP in order to develop a novel detection method or improve crop management.

Identification of Oil Palm's Consistently Upregulated Genes during Early Infections of Ganoderma boninense via RNA-Seq Technology and Real-Time Quantitative PCR.

Basal stem rot (BSR) disease caused by pathogenic fungus Ganoderma boninense is a significant concern in the oil palm industry. G. boninense infection in oil palm induces defense-related genes. To understand oil palm defense mechanisms in response to fungal invasion, we analyzed differentially expressed genes (DEGs) derived from RNA-sequencing (RNA-seq) transcriptomic libraries of oil palm roots infected with G. boninense. A total of 126 DEGs were detected from the transcriptomic libraries of G. boninense-infected root tissues at different infection stages. Functional annotation via pathway enrichment analyses revealed that the DEGs were involved in the defense response against the pathogen. The expression of the selected DEGs was further confirmed using real-time quantitative PCR (qPCR) on independent oil palm seedlings and mature palm samples. Seven putative defense-related DEGs consistently showed upregulation in seedlings and mature plants during G. boninense infection. These seven genes might potentially be developed as biomarkers for the early detection of BSR in oil palm.

Early nodulin 93 protein gene: essential for induction of somatic embryogenesis in oil palm.

KEY MESSAGE: Transcript profiling during the early induction phase of oil palm tissue culture and RNAi studies in a model somatic embryogenesis system showed that EgENOD93 expression is essential for somatic embryogenesis. Micropropagation of oil palm through tissue culture is vital for the generation of superior and uniform elite planting materials. Studies were carried out to identify genes to distinguish between leaf explants with the potential to develop into embryogenic or non-embryogenic callus. Oil palm cDNA microarrays were co-hybridized with cDNA probes of reference tissue, separately with embryo forming (media T527) and non-embryo (media T694) forming leaf explants sampled at Day 7, Day 14 and Day 21. Analysis of the normalized datasets has identified 77, 115 and 127 significantly differentially expressed genes at Day 7, Day 14, and Day 21, respectively. An early nodulin 93 protein gene (ENOD93), was highly expressed at Day 7, Day 14, and Day 21 and in callus (media T527), as assessed by RT-qPCR. Validation of EgENOD93 across tissue culture lines of different genetic background and media composition showed the potential of this gene as an embryogenic marker. In situ RNA hybridization and functional characterization in Medicago truncatula provided additional evidence that ENOD93 is essential for somatic embryogenesis. This study supports the suitability of EgENOD93 as a marker to predict the potential of leaf explants to produce embryogenic callus. Crosstalk among stresses, auxin, and Nod-factor like signalling molecules likely induces the expression of EgENOD93 for embryogenic callus formation.

Physical and Physiological Monitoring on Red Palm Weevil-Infested Oil Palms.

The red palm weevil (RPW) is a stem boring Coleoptera that decimates host palm trees from within. The challenge of managing this pest is due to a lack of physical symptoms during the early stages of infestation. Investigating the physiological changes that occur within RPW-infested palm trees may be useful in establishing a new approach in RPW detection. In this study, the effects of RPW infestation were investigated in Elaeis guineensis by observing changes in physical and physiological parameters during the progress of infestation by visual inspection and the comparison of growth, gas exchange, stomatal conductance, and chlorophyll content between the non-infested control, physically wounded, and RPW-infested E. guineensis groups. During the study period, four distinct levels of physical infestation were observed and recorded. The RPW-infested group displayed significantly lower maximum photosynthesis activity (Amax) starting from the third week post-infestation. However, growth in terms of change in plant height and stem circumference, leaves' stomatal conductance, and chlorophyll content were not significantly different between the three groups during the duration of the study. The significant drop in photosynthesis was observed one week before physical changes appeared. This suggests the promising utilisation of photosynthesis activity as a signal for detecting RPW infestation at the early stage of attacks, which could be useful for integration in integrated pest management (IPM).

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

Postharvest analysis of lowland transgenic tomato fruits harboring hpRNAi-ACO1 construct.

The plant hormone, ethylene, is an important regulator which involved in regulating fruit ripening and flower senescence. In this study, RNA interference (RNAi) technology was employed to silence the genes involved in ethylene biosynthetic pathway. This was achieved by blocking the expression of specific gene encoding the ACC oxidase. Initially, cDNA corresponding to ACO1 of lowland tomato cultivar (MT1), which has high identity with ACO1 of Solanum lycopersicum in GenBank, was cloned through RT-PCR. Using a partial coding region of ACO1, one hpRNAi transformation vector was constructed and expressed ectopically under the 35S promoter. Results showed that transgenic lines harboring the hpRNA-ACO1 construct had lower ethylene production and a longer shelf life of 32 days as compared to 10 days for wild-type fruits. Changes in cell wall degrading enzyme activities were also investigated in cases where the transgenic fruits exhibited reduced rates of firmness loss, which can be associated with a decrease in pectin methylesterase (PME) and polygalacturonase (PG) activities. However, no significant change was detected in both transgenic and wild-type fruits in terms of ß-galactosidase (ß-Gal) activity and levels of total soluble solid, titratable acid and ascorbic acid.

Effects of 1-methylcyclopropene and modified atmosphere packaging on the antioxidant capacity in pepper "Kulai" during low-temperature storage.

The objective of the present study was to simultaneously evaluate the effect of a postharvest treatment on the pepper's antioxidant content and its ability to retain its economical value during the postharvest period. The fruits were pretreated by modified atmosphere packaging (MAP) with or without treatment with 1-methylcyclopropene (1-MCP) before cold storage at 10°C. Changes in the levels of non-enzymatic antioxidants, including the total phenolic, ascorbic acid levels and the total glutathione level, as well as enzymatic antioxidants, including ascorbate peroxidase (APX), glutathione reductase (GR), and catalase (CAT), were determined. Both treatments successfully extended the shelf life of the fruit for up to 25 days, and a high level of antioxidant capacity was maintained throughout the storage period. However, 1-MCP treatment maintained the high antioxidant capacity for a longer period of time. The 1-MCP-treated peppers maintained high levels of phenolic content, a high reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio, decreased levels of ascorbic acid and CAT activity, and increased levels of APX and GR compared with the peppers that were not treated with 1-MCP. The overall results suggested that a combination of 1-MCP and MAP was the most effective treatment for extending shelf life while retaining the nutritional benefits.

The stearoyl-acyl-carrier-protein desaturase promoter (Des) from oil palm confers fruit-specific GUS expression in transgenic tomato.

The stearoyl-acyl-carrier-protein (ACP) desaturase is a plastid-localized enzyme that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays an important role in the determination of the properties of the majority of cellular glycerolipids. Functional characterization of the fatty acid desaturase genes and their specific promoters is a prerequisite for altering the composition of unsaturated fatty acids of palm oil by genetic engineering. In this paper, the specificity and strength of the oil palm stearoyl-ACP desaturase gene promoter (Des) was evaluated in transgenic tomato plants. Transcriptional fusions between 5' deletions of the Des promoter (Des1-4) and the ß-glucuronidase (GUS) reporter gene were generated and their expression analyzed in different tissues of stably transformed tomato plants. Histochemical analysis of the Des promoter deletion series revealed that GUS gene expression was confined to the tomato fruits. No expression was detected in vegetative tissues of the transgenic plants. The highest levels of GUS activity was observed in different tissues of ripe red fruits (vascular tissue, septa, endocarp, mesocarp and columella) and in seeds, which harbored the promoter region located between -590 and +10. A comparison of the promoter-deletion constructs showed that the Des4 promoter deletion (314bp) produced a markedly low level of GUS expression in fruits and seeds. Fluorometric analysis of the GUS activity revealed a 4-fold increase in the activity of the full-length Des promoter compared to the CaMV35S promoter. RNA-hybridization analyses provided additional evidence of increased GUS expression in fruits driven by a Des fragment. Taken together, these results demonstrate the potential of the Des promoter as a tool for the genetic engineering of oil palms and other species, including dicots, in improving the quality and nutritional value of the fruits.