Diverse and dynamic roles of F-box proteins in plant biology.

The SCF complex is a widely studied multi-subunit ring E3 ubiquitin ligase that tags targeted proteins with ubiquitin for protein degradation by the ubiquitin 26S-proteasome system (UPS). The UPS is an important system that generally keeps cellular events tightly regulated by purging misfolded or damaged proteins and selectively degrading important regulatory proteins. The specificity of this post-translational regulation is controlled by F-box proteins (FBPs) via selective recognition of a protein-protein interaction motif at the C-terminal domain. Hence, FBPs are pivotal proteins in determining the plant response in multiple scenarios. It is not surprising that the FBP family is one of the largest protein families in the plant kingdom. In this review, the roles of FBPs, specifically in plants, are compiled to provide insights into their involvement in secondary metabolites, plant stresses, phytohormone signalling, plant developmental processes and miRNA biogenesis.

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.

Green leaf volatiles: biosynthesis, biological functions and their applications in biotechnology.

Plants have evolved numerous constitutive and inducible defence mechanisms to cope with biotic and abiotic stresses. These stresses induce the expression of various genes to activate defence-related pathways that result in the release of defence chemicals. One of these defence mechanisms is the oxylipin pathway, which produces jasmonates, divinylethers and green leaf volatiles (GLVs) through the peroxidation of polyunsaturated fatty acids (PUFAs). GLVs have recently emerged as key players in plant defence, plant-plant interactions and plant-insect interactions. Some GLVs inhibit the growth and propagation of plant pathogens, including bacteria, viruses and fungi. In certain cases, GLVs released from plants under herbivore attack can serve as aerial messengers to neighbouring plants and to attract parasitic or parasitoid enemies of the herbivores. The plants that perceive these volatile signals are primed and can then adapt in preparation for the upcoming challenges. Due to their 'green note' odour, GLVs impart aromas and flavours to many natural foods, such as vegetables and fruits, and therefore, they can be exploited in industrial biotechnology. The aim of this study was to review the progress and recent developments in research on the oxylipin pathway, with a specific focus on the biosynthesis and biological functions of GLVs and their applications in industrial biotechnology.

Functional characterization of sesquiterpene synthase from Polygonum minus.

Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of ß -sesquiphellandrene.

Transcriptome profiling of genes induced by salicylic acid and methyl jasmonate in Polygonum minus.

The importance of plant secondary metabolites for both mankind and the plant itself has long been established. However, despite extensive research on plant secondary metabolites, plant secondary metabolism and its regulation still remained poorly characterized. In this present study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript profiling was applied to generate the expression profiles of Polygonum minus in response to salicylic acid (SA) and methyl jasmonate (MeJA) elicitations. This study reveals two different sets of genes induced by SA and MeJA, respectively where stress-related genes were proved to lead to the expression of genes involved in plant secondary metabolite biosynthetic pathways. A total of 98 transcript-derived fragments (TDFs) were up-regulated, including 46 from SA-treated and 52 from MeJA-treated samples. The cDNA-AFLP transcripts generated using 64 different Mse1/Taq1 primer combinations showed that treatments with SA and MeJA induced genes mostly involved in scavenging reactive oxygen species, including zeaxanthin epoxidase, cytosolic ascorbate peroxidase 1 and peroxidase. Of these stress-related genes, 15 % of other annotated TDFs are involved mainly in secondary metabolic processes where among these, two genes encoding (+)-delta cadinene synthase and cinnamoyl-CoA reductase were highlighted.

Induction and analysis of the alkaloid mitragynine content of a Mitragyna speciosa suspension culture system upon elicitation and precursor feeding.

This study aimed to determine the effects of different concentrations and combinations of the phytohormones 2,4-dichlorophenoxy acetic acid (2,4-D), kinetin, 6-benzylaminopurine (BAP), and 1-naphthaleneacetic acid (NAA) on callus induction and to demonstrate the role of elicitors and exogenous precursors on the production of mitragynine in a Mitragyna speciosa suspension culture. The best callus induction was achieved from petiole explants cultured on WPM that was supplemented with 4 mg L⁻¹ 2,4-D (70.83%). Calli were transferred to liquid media and agitated on rotary shakers to establish Mitragyna speciosa cell suspension cultures. The optimum settled cell volume was achieved in the presence of WPM that contained 3 mg L⁻¹ 2,4-D and 3% sucrose (9.47 ± 0.4667 mL). The treatment of cultures with different concentrations of yeast extract and salicylic acid for different inoculation periods revealed that the highest mitragynine content as determined by HPLC was achieved from the culture treated with 250 mg L⁻¹ yeast extract (9.275 ± 0.082 mg L⁻¹) that was harvested on day 6 of culturing; salicylic acid showed low mitragynine content in all concentrations used. Tryptophan and loganin were used as exogenous precursors; the highest level of mitragynine production was achieved in cultures treated with 3 µM tryptophan and harvested at 6 days (13.226 ± 1.98 mg L⁻¹).

Overview of Repressive miRNA Regulation by Short Tandem Target Mimic (STTM): Applications and Impact on Plant Biology.

The application of miRNA mimic technology for silencing mature miRNA began in 2007. This technique originated from the discovery of the INDUCED BY PHOSPHATE STARVATION 1 (IPS1) gene, which was found to be a competitive mimic that prevents the cleavage of the targeted mRNA by miRNA inhibition at the post-transcriptional level. To date, various studies have been conducted to understand the molecular mimic mechanism and to improve the efficiency of this technology. As a result, several mimic tools have been developed: target mimicry (TM), short tandem target mimic (STTM), and molecular sponges (SPs). STTM is the most-developed tool due to its stability and effectiveness in decoying miRNA. This review discusses the application of STTM technology on the loss-of-function studies of miRNA and members from diverse plant species. A modified STTM approach for studying the function of miRNA with spatial-temporal expression under the control of specific promoters is further explored. STTM technology will enhance our understanding of the miRNA activity in plant-tissue-specific development and stress responses for applications in improving plant traits via miRNA regulation.

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.

Flavonoid biosynthesis genes putatively identified in the aromatic plant Polygonum minus via Expressed Sequences Tag (EST) analysis.

P. minus is an aromatic plant, the leaf of which is widely used as a food additive and in the perfume industry. The leaf also accumulates secondary metabolites that act as active ingredients such as flavonoid. Due to limited genomic and transcriptomic data, the biosynthetic pathway of flavonoids is currently unclear. Identification of candidate genes involved in the flavonoid biosynthetic pathway will significantly contribute to understanding the biosynthesis of active compounds. We have constructed a standard cDNA library from P. minus leaves, and two normalized full-length enriched cDNA libraries were constructed from stem and root organs in order to create a gene resource for the biosynthesis of secondary metabolites, especially flavonoid biosynthesis. Thus, large-scale sequencing of P. minus cDNA libraries identified 4196 expressed sequences tags (ESTs) which were deposited in dbEST in the National Center of Biotechnology Information (NCBI). From the three constructed cDNA libraries, 11 ESTs encoding seven genes were mapped to the flavonoid biosynthetic pathway. Finally, three flavonoid biosynthetic pathway-related ESTs chalcone synthase, CHS (JG745304), flavonol synthase, FLS (JG705819) and leucoanthocyanidin dioxygenase, LDOX (JG745247) were selected for further examination by quantitative RT-PCR (qRT-PCR) in different P. minus organs. Expression was detected in leaf, stem and root. Gene expression studies have been initiated in order to better understand the underlying physiological processes.

Data on transcriptome analysis from mesocarp tissue of mango Mangifera indica 'Chokanan' fruits.

Mangoes comes in different sizes and consumer are often favour those with bigger, fleshy mango. Many genes are plays an important role in determining the growth, final size and shapes of the mango. To further understand the roles of genes that play roles in fruit development, a de novo transcriptomic analysis was performed at two stages of fruit development; immature and ripening stage, using Illumina HiSeq 4000 platform with 30× sequencing coverage. A total of approximately 128 Gb of clean nucleotides was obtained from 130 Gb of raw nucletides sequenced from four fruit mesocarp of both time points. The raw and clean data were deposited into National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database with accession number PRJNA803945.

A Review of Integrative Omic Approaches for Understanding Rice Salt Response Mechanisms.

Soil salinity is one of the most serious environmental challenges, posing a growing threat to agriculture across the world. Soil salinity has a significant impact on rice growth, development, and production. Hence, improving rice varieties' resistance to salt stress is a viable solution for meeting global food demand. Adaptation to salt stress is a multifaceted process that involves interacting physiological traits, biochemical or metabolic pathways, and molecular mechanisms. The integration of multi-omics approaches contributes to a better understanding of molecular mechanisms as well as the improvement of salt-resistant and tolerant rice varieties. Firstly, we present a thorough review of current knowledge about salt stress effects on rice and mechanisms behind rice salt tolerance and salt stress signalling. This review focuses on the use of multi-omics approaches to improve next-generation rice breeding for salinity resistance and tolerance, including genomics, transcriptomics, proteomics, metabolomics and phenomics. Integrating multi-omics data effectively is critical to gaining a more comprehensive and in-depth understanding of the molecular pathways, enzyme activity and interacting networks of genes controlling salinity tolerance in rice. The key data mining strategies within the artificial intelligence to analyse big and complex data sets that will allow more accurate prediction of outcomes and modernise traditional breeding programmes and also expedite precision rice breeding such as genetic engineering and genome editing.

Genome-wide analysis of sulfur-encoding biosynthetic genes in rice (Oryza sativa L.) with Arabidopsis as the sulfur-dependent model plant.

Sulfur is an essential element required for plant growth and development, physiological processes and stress responses. Sulfur-encoding biosynthetic genes are involved in the primary sulfur assimilation pathway, regulating various mechanisms at the gene, cellular and system levels, and in the biosynthesis of sulfur-containing compounds (SCCs). In this study, the SCC-encoding biosynthetic genes in rice were identified using a sulfur-dependent model plant, the Arabidopsis. A total of 139 AtSCC from Arabidopsis were used as reference sequences in search of putative rice SCCs. At similarity index > 30%, the similarity search against Arabidopsis SCC query sequences identified 665 putative OsSCC genes in rice. The gene synteny analysis showed a total of 477 syntenic gene pairs comprised of 89 AtSCC and 265 OsSCC biosynthetic genes in Arabidopsis and rice, respectively. Phylogenetic tree of the collated (AtSCCs and OsSCCs) SCC-encoding biosynthetic genes were divided into 11 different clades of various sizes comprised of branches of subclades. In clade 1, nearing equal representation of OsSCC and AtSCC biosynthetic genes imply the most ancestral lineage. A total of 25 candidate Arabidopsis SCC homologs were identified in rice. The gene ontology enrichment analysis showed that the rice-Arabidopsis SCC homologs were significantly enriched in the following terms at false discovery rate (FDR) < 0.05: (i) biological process; sulfur compound metabolic process and organic acid metabolic processes, (ii) molecular function; oxidoreductase activity, acting on paired donors with incorporation or reduction of molecular oxygen and (iii) KEGG pathway; metabolic pathways and biosynthesis of secondary metabolites. At less than five duplicated blocks of separation, no tandem duplications were observed among the SCC biosynthetic genes distributed in rice chromosomes. The comprehensive rice SCC gene description entailing syntenic events with Arabidopsis, motif distribution and chromosomal mapping of the present findings offer a foundation for rice SCC gene functional studies and advanced strategic rice breeding.

Multi-Omics Approaches and Resources for Systems-Level Gene Function Prediction in the Plant Kingdom.

In higher plants, the complexity of a system and the components within and among species are rapidly dissected by omics technologies. Multi-omics datasets are integrated to infer and enable a comprehensive understanding of the life processes of organisms of interest. Further, growing open-source datasets coupled with the emergence of high-performance computing and development of computational tools for biological sciences have assisted in silico functional prediction of unknown genes, proteins and metabolites, otherwise known as uncharacterized. The systems biology approach includes data collection and filtration, system modelling, experimentation and the establishment of new hypotheses for experimental validation. Informatics technologies add meaningful sense to the output generated by complex bioinformatics algorithms, which are now freely available in a user-friendly graphical user interface. These resources accentuate gene function prediction at a relatively minimal cost and effort. Herein, we present a comprehensive view of relevant approaches available for system-level gene function prediction in the plant kingdom. Together, the most recent applications and sought-after principles for gene mining are discussed to benefit the plant research community. A realistic tabulation of plant genomic resources is included for a less laborious and accurate candidate gene discovery in basic plant research and improvement strategies.

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.

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.

Application of Reverse Genetics in Functional Genomics of Potyvirus.

Numerous potyvirus studies, including virus biology, transmission, viral protein function, as well as virus-host interaction, have greatly benefited from the utilization of reverse genetic techniques. Reverse genetics of RNA viruses refers to the manipulation of viral genomes, transfection of the modified cDNAs into cells, and the production of live infectious progenies, either wild-type or mutated. Reverse genetic technology provides an opportunity of developing potyviruses into vectors for improving agronomic traits in plants, as a reporter system for tracking virus infection in hosts or a production system for target proteins. Therefore, this review provides an overview on the breakthroughs achieved in potyvirus research through the implementation of reverse genetic systems.

Sequence and Phylogenetic Analysis of the First Complete Genome of Ricetungro spherical virus in Malaysia.

BACKGROUND: Rice tungro disease (RTD) is a viral disease mainly affecting rice in Asia. RTD caused by Rice tungro bacilliform virus and Rice tungro spherical virus. To date, there are only 5 RTSV isolates have been reported. OBJECTIVES: In this study, we aimed to report the complete nucleotide sequence of Malaysian isolate of Rice tungro spherical virus Seberang Perai (RTSV-SP) for the first time. RTSV-SP was characterized and its evolutionary relationship with previously reported Indian and Philippines isolates were elucidated. MATERIALS AND METHODS: RTSV-SP isolate was isolated from a recent outbreak in a paddy field in Seberang Perai zone of Malaysia. Its complete genome was amplified by RT-PCR, cloned and sequenced. RESULTS: Sequence analysis indicated that the genome of RTSV-SP consisted of 12,173 nucleotides (nt). Comparative analysis of 6 complete genome sequences using Clustal Omega showed that Seberang Perai isolate shared the highest nucleotide identity (96.04%) with Philippine-A isolate, except that the sORF-2 of RTSV-SP is shorter than RTSV Philippine-A by 27 amino acid residues. RTSV-SP found to cluster in Southeast Asia (SEA) group based on the whole genome sequence phylogenetic analysis using MEGA X software. CONCLUSIONS: Phylogenetic classification of RTSV isolates based on the complete nucleotide sequences showed more distinctive clustering pattern with the addition of RTSV-SP whole genome to the available isolates. Present study described the isolation and molecular characterization of RTSV-SP.

Data on genome resequencing of pigmented and non-pigmented Malaysian rice varieties.

The genomics and genetic data of pigmented and non-pigmented Malaysian rice varieties are still limited. Hence, we performed the genome resequencing of two black rice varieties (Bali, Pulut Hitam 9), two red rice varieties (MRM16, MRQ100) and two white rice varieties (MR297 and MRQ76) using Illumina HiSeq 4000 platform with 30x sequencing coverage. We aimed to identify and annotate single nucleotide polymorphisms (SNPs) from the genome of these four pigmented and two non-pigmented rice varieties. The potential SNPs will be used in developing the functional SNP markers related to nutritional (i.e. antioxidant, folate, amylose) and quality (i.e. aromatic) traits. Raw data of the pigmented and non-pigmented rice varieties have been deposited into the European Nucleotide Archive (ENA) database with accession number PRJEB29070 and PRJEB32344, respectively.

RNA-seq data from whole rice grains of pigmented and non-pigmented Malaysian rice varieties.

Pigmented rice is enriched with antioxidants, macro- and micronutrients. A comprehensive investigation of the gene expression patterns among the pigmented rice varieties would help to understand the cellular mechanism and biological processes of rice grain pigmentation. Hence, we performed RNA sequencing and analysis on the whole grain of dehusked mature seeds of selected six Malaysian rice varieties with varying grain pigmentations. These varieties were black rice (BALI and Pulut Hitam 9), red rice (MRM16 and MRQ100) and white rice (MR297 and MRQ76). Illumina HiSeq™ 4000 sequencer was used to generate total raw nucleotides of approximately 53 Gb in size. From 353,937,212 total paired-end raw reads, 340,131,496 total clean reads were obtained. The raw reads were deposited into European Nucleotide Archive (ENA) database and can be accessed via accession number PRJEB34340. This dataset allows us to identify and profile all expressed genes with functions related to nutritional traits (i.e. antioxidants, folate and amylose content) and quality trait (i.e. aroma) across both pigmented and non-pigmented rice varieties. In addition, the transcriptome data obtained will be valuable for discovery of potential gene markers and functional SNPs related to functional traits to assist in rice breeding programme.