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HnRNP-like proteins as post-transcriptional regulators

Yeap WC, Namasivayam P, Ho CL

Plant Sci, 2014 Oct;227:90-100.
PMID: 25219311 DOI: 10.1016/j.plantsci.2014.07.005

Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses.
Fulltext An Efficient Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Protein 9 Mutagenesis System for Oil Palm (Elaeis guineensis)

Yeap WC, Norkhairunnisa Che Mohd Khan, Norfadzilah Jamalludin, Muad MR, Appleton DR, Harikrishna Kulaveerasingam

Front Plant Sci, 2021;12:773656.
PMID: 34880893 DOI: 10.3389/fpls.2021.773656

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful tool for the precise editing of plant genomes for crop improvement. Rapid in vitro methods for the determination of guide RNA (gRNA) cleavage efficiency and an efficient DNA delivery system is essential for gene editing. However, we lack an efficient gene-editing system for palm species. In this study, we described the development of a transient oil palm protoplast assay to rapidly evaluate the cleavage efficiency of CRISPR/Cas9 mutagenesis and the generation of stable transformed oil palms using biolistic particle bombardment in immature embryos. Using the phytoene desaturase (EgPDS) gene, we found cleavage frequency of up to 25.49% in electro-transfected protoplast, which enables the production of transgenic oil palm shoots exhibiting chimeric albino phenotypes as a result of DNA insertions, deletions (InDels), and nucleotide substitutions, with a mutation efficiency of 62.5-83.33%. We further validated the mutagenesis efficiency and specificity of the CRISPR/Cas9 system in oil palm by targeting the brassinosteroid-insensitive 1 (EgBRI1) gene, which resulted in nucleotide substitutions in EgBRI1 with premature necrosis phenotype in oil palm transgenic shoots and stunted phenotype resulting from DNA InDels. Taken together, our results showed that effective and efficient editing of genes using the CRISPR/Cas9 system can be achieved in oil palm by optimizing the selection of efficient gRNA and DNA delivery methods. This newly designed strategy will enable new routes for the genetic improvement in oil palm and related species.
EgRBP42 from oil palm enhances adaptation to stress in Arabidopsis through regulation of nucleocytoplasmic transport of stress-responsive mRNAs

Yeap WC, Namasivayam P, Ooi TEK, Appleton DR, Kulaveerasingam H, Ho CL

Plant Cell Environ, 2019 05;42(5):1657-1673.
PMID: 30549047 DOI: 10.1111/pce.13503

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.
EgRBP42 encoding an hnRNP-like RNA-binding protein from Elaeis guineensis Jacq. is responsive to abiotic stresses

Yeap WC, Ooi TE, Namasivayam P, Kulaveerasingam H, Ho CL

Plant Cell Rep, 2012 Oct;31(10):1829-43.
PMID: 22699852 DOI: 10.1007/s00299-012-1297-x

RNA-binding proteins (RBPs) have been implicated as regulatory proteins involved in the post-transcriptional processes of gene expression in plants under various stress conditions. In this study, we report the cloning and characterization of a gene, designated as EgRBP42, encoding a member of the plant heterogeneous nuclear ribonucleoprotein (hnRNP)-like RBP family from oil palm (Elaeis guineensis Jacq.). EgRBP42 consists of two N-terminal RNA recognition motifs and a glycine-rich domain at the C-terminus. The upstream region of EgRBP42 has multiple light-responsive, stress-responsive regulatory elements and regulatory elements associated with flower development. Real-time RT-PCR analysis of EgRBP42 showed that EgRBP42 was expressed in oil palm tissues tested, including leaf, shoot apical meristem, root, female inflorescence, male inflorescence and mesocarp with the lowest transcript level in the roots. EgRBP42 protein interacted with transcripts associated with transcription, translation and stress responses using pull-down assay and electrophoretic mobility shift assay. The accumulation of EgRBP42 and its interacting transcripts were induced by abiotic stresses, including salinity, drought, submergence, cold and heat stresses in leaf discs. Collectively, the data suggested that EgRBP42 is a RBP, which responds to various abiotic stresses and could be advantageous for oil palm under stress conditions. Key message EgRBP42 may be involved in the post-transcriptional regulation of stress-related genes important for plant stress response and adaptation.
WRI1-1, ABI5, NF-YA3 and NF-YC2 increase oil biosynthesis in coordination with hormonal signaling during fruit development in oil palm

Yeap WC, Lee FC, Shabari Shan DK, Musa H, Appleton DR, Kulaveerasingam H

Plant J, 2017 Jul;91(1):97-113.
PMID: 28370622 DOI: 10.1111/tpj.13549

The oil biosynthesis pathway must be tightly controlled to maximize oil yield. Oil palm accumulates exceptionally high oil content in its mesocarp, suggesting the existence of a unique fruit-specific fatty acid metabolism transcriptional network. We report the complex fruit-specific network of transcription factors responsible for modulation of oil biosynthesis genes in oil palm mesocarp. Transcriptional activation of EgWRI1-1 encoding a key master regulator that activates expression of oil biosynthesis genes, is activated by three ABA-responsive transcription factors, EgNF-YA3, EgNF-YC2 and EgABI5. Overexpression of EgWRI1-1 and its activators in Arabidopsis accelerated flowering, increased seed size and oil content, and altered expression levels of oil biosynthesis genes. Protein-protein interaction experiments demonstrated that EgNF-YA3 interacts directly with EgWRI1-1, forming a transcription complex with EgNF-YC2 and EgABI5 to modulate transcription of oil biosynthesis pathway genes. Furthermore, EgABI5 acts downstream of EgWRKY40, a repressor that interacts with EgWRKY2 to inhibit the transcription of oil biosynthesis genes. We showed that expression of these activators and repressors in oil biosynthesis can be induced by phytohormones coordinating fruit development in oil palm. We propose a model highlighting a hormone signaling network coordinating fruit development and fatty acid biosynthesis.
Fulltext Comparative transcriptome analysis reveals novel insights into transcriptional responses to phosphorus starvation in oil palm (Elaeis guineensis) root

Kong SL, Abdullah SNA, Ho CL, Musa MHB, Yeap WC

BMC Genom Data, 2021 02 05;22(1):6.
PMID: 33568046 DOI: 10.1186/s12863-021-00962-7

BACKGROUND: Phosphorus (P), in its orthophosphate form (Pi) is an essential macronutrient for oil palm early growth development in which Pi deficiency could later on be reflected in lower biomass production. Application of phosphate rock, a non-renewable resource has been the common practice to increase Pi accessibility and maintain crop productivity in Malaysia. However, high fixation rate of Pi in the native acidic tropical soils has led to excessive utilization of P fertilizers. This has caused serious environmental pollutions and cost increment. Even so, the Pi deficiency response mechanism in oil palm as one of the basic prerequisites for crop improvement remains largely unknown.
RESULTS: Using total RNA extracted from young roots as template, we performed a comparative transcriptome analysis on oil palm responding to 14d and 28d of Pi deprivation treatment and under adequate Pi supply. By using Illumina HiSeq4000 platform, RNA-Seq analysis was successfully conducted on 12 paired-end RNA-Seq libraries and generated more than 1.2 billion of clean reads in total. Transcript abundance estimated by fragments per kilobase per million fragments (FPKM) and differential expression analysis revealed 36 and 252 genes that are differentially regulated in Pi-starved roots at 14d and 28d, respectively. Genes possibly involved in regulating Pi homeostasis, nutrient uptake and transport, hormonal signaling and gene transcription were found among the differentially expressed genes.
CONCLUSIONS: Our results showed that the molecular response mechanism underlying Pi starvation in oil palm is complexed and involved multilevel regulation of various sensing and signaling components. This contribution would generate valuable genomic resources in the effort to develop oil palm planting materials that possess Pi-use efficient trait through molecular manipulation and breeding programs.
Fulltext Differential abundance analysis of mesocarp protein from high- and low-yielding oil palms associates non-oil biosynthetic enzymes to lipid biosynthesis

Ooi TE, Yeap WC, Daim LD, Ng BZ, Lee FC, Othman AM, et al.

Proteome Sci, 2015;13:28.
PMID: 26617468 DOI: 10.1186/s12953-015-0085-2

BACKGROUND: The oil palm Elaeis guineensis Jacq. which produces the highest yield per unit land area of the oil crops is the most important commercial oil crop in South East Asia. The fleshy mesocarp of oil palm fruit, where oil is mostly derived from, contains up to 90 % dry weight of oil (one of the most concentrated in plant tissues). Hence, there is attention given to gain insights into the processes of oil deposition in this oil rich tissue. For that purpose, two-dimensional differential gel electrophoresis (DIGE) coupled with western assays, were used here to analyze differential protein levels in genetically-related high-and low-yielding oil palm mesocarps.
RESULTS: From the DIGE comparative analysis in combination with western analysis, 41 unique differentially accumulated proteins were discovered. Functional categorization of these proteins placed them in the metabolisms of lipid, carbohydrate, amino acids, energy, structural proteins, as well as in other functions. In particular, higher abundance of fructose-1,6-biphosphate aldolase combined with reduced level of triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase may be indicative of important flux balance changes in glycolysis, while amino acid metabolism also appeared to be closely linked with oil yield.
CONCLUSIONS: Forty-one proteins in several important biological pathways were identified as exhibiting differential in abundance at critical oil production stages. These confirm that oil yield is a complex trait involving the regulation of genes in multiple biological pathways. The results also provide insights into key control points of lipid biosynthesis in oil palm and can assist in the development of genetic markers for use in oil palm breeding programmes.