Zamri Zainal

MicroRNA and transcription factor: Key players in plant regulatory network

Recent achievements in plant microRNA (miRNA), a large class of small and non-coding RNAs, are very exciting. A wide array of techniques involving forward genetic, molecular cloning, bioinformatic analysis, and the latest technology, deep sequencing have greatly advanced miRNA discovery. A tiny miRNA sequence has the ability to target single/multiple mRNA targets. Most of the miRNA targets are transcription factors (TFs) which have paramount importance in regulating the plant growth and development. Various families of TFs, which have regulated a range of regulatory networks, may assist plants to grow under normal and stress environmental conditions. This present review focuses on the regulatory relationships between miRNAs and different families of TFs like; NF-Y, MYB, AP2, TCP, WRKY, NAC, GRF, and SPL. For instance NF-Y play important role during drought tolerance and flower development, MYB are involved in signal transduction and biosynthesis of secondary metabolites, AP2 regulate the floral development and nodule formation, TCP direct leaf development and growth hormones signaling. WRKY have known roles in multiple stress tolerances, NAC regulate lateral root formation, GRF are involved in root growth, flower, and seed development, and SPL regulate plant transition from juvenile to adult. We also studied the relation between miRNAs and TFs by consolidating the research findings from different plant species which will help plant scientists in understanding the mechanism of action and interaction between these regulators in the plant growth and development under normal and stress environmental conditions.

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.

Identification of cDNAs for jasmonic acid-responsive genes in Polygonum minus roots by suppression subtractive hybridization

Elicitation, the plant-based biotechnology approach that utilizes the ability of plant roots to absorb and secrete a vast variety of bioactive compounds, was studied on Polygonum minus using jasmonic acid (JA) as an elicitor. To understand the overall molecular responses of P. minus roots to JA induction, a subtracted cDNA library was constructed using the suppression subtractive hybridization (SSH) method. From a total of 1,344 randomly selected colonies, 190 clones were shown to be differentially expressed using Reverse Northern hybridization. BLAST analysis revealed that clones were similar to genes associated with the biosynthesis of aromatic compounds through the oxylipin pathway, such as alcohol dehydrogenase and lipoxygenase. Putative clones involved in the shikimate pathway, including S-adenosyl-l-methionine synthetase and S-adenosyl-l-homocysteine hydrolase, were identified with predicted roles in phenylpropanoids’ biosynthesis. Genes responding to abiotic stress unique to JA elicitation, such as ELI3-1, glutathione S-transferase and peroxidase 1, were also identified. The kelch-repeat containing F-box family protein, a possible transcription factor in response to JA elicitation was also found. The results of the RT-PCR showed that the eight selected clones were strongly up-regulated, except for lipoxygenase, which showed a slightly higher expression of the transcript levels in response to the JA elicitation.

RNA-seq analysis for secondary metabolite pathway gene discovery in Polygonum minus

Polygonum minus plant is rich in secondary metabolites, especially terpenoids and flavonoids. Present study generates transcriptome resource for P. minus to decipher its secondary metabolite biosynthesis pathways. Raw reads and the transcriptome assembly project have been deposited at GenBank under the accessions SRX313492 (root) and SRX669305 (leaf) respectively.

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 β-galactosidase (β-Gal) activity and levels of total soluble solid, titratable acid and ascorbic acid.

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.

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 β-sesquiphellandrene.

Molecular cloning and characterization of strictosidine synthase, a key gene in biosynthesis of mitragynine from Mitragyna speciosa

Mitragynine is one of the most dominant indole alkaloids present in the leaves of Mitragyna speciosa , a species of Rubiaceae. This alkaloid is believed to be synthesized via condensation of the amino acid derivative, tryptamine and secologanine by the action of strictosidine synthase (STR). The cDNA clone encoding STR from M. specios a was cloned through reverse-transcription polymerase chain reaction (RT-PCR) and denoted as StrMs1. The clone is a full-length cDNA with a size of 1257 bp, which contains an open reading frame of 1056 bp starting from base pair 18 to 1076. Sequence analysis showed that StrMs1 has high homology with other STRs of TIA-producing plants. Nucleotide sequence of StrMs1 was deposited in GenBank with accession number ADK91432. The deduced amino acid sequence has 352 residues with a predicted molecular weight of 39 kDa and isoelectric point at pH 5.78. Southern blot performed showed that there is only one copy of StrMs1 present in the genome of M. speciosa . Expression pattern on different tissues tested using RT-PCR revealed that besides leaf, the expression was also detected in root, stem and flower. Expression profiles under plant defense signal using salicylic acid (SA) was investigated on leaf tissues and the results showed that the transcript of StrMs1 were detected before and after treatment with salicylic acid. Result obtained from phylogenetic analysis suggested that StrMs1 is the most evolved protein among other STRs. However, the 3-D prediction of StrMs1 showed that there are alpha helices and beta propeller structures, which remain conserved withother STRs. Key word: Strictosidine synthase, Mitragyna speciosa, StrMs1, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), molecular evolution, protein prediction.

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).

Alteration of Abiotic Stress Responsive Genes in Polygonum minus Roots by Jasmonic Acid Elicitation

Plants are continuously exposed to both biotic and abiotic stress in their natural environment. Unlike animals, plants are immobilized organisms which tend to be vulnerable to various environmental stresses. In order to survive, plants have evolved a wide range of defense mechanism to cope with these stresses. Both biotic and abiotic stresses might share some common signaling pathway in triggering the defense system in plants. Recent researches have revealed that phytohormones such as abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) are intermediate molecules which play key roles in the crosstalk between biotic and abiotic signaling network (Fujita et al. 2006). In this chapter, we highlight the effects of exogenous applied jasmonic acid in triggering the synthesis of some molecules and activating their respective biosynthetic genes in plants as a response towards abiotic stresses. Abiotic stress is defined as non-living external factors, usually environment conditions, which could reduce plant growth and cause huge devastation on agricultural productivity. Some of these major adverse environmental factors are drought, salinity, heavy metals, extreme temperatures, nutrient poor soils and other source of natural disasters. To our knowledge, these abiotic stresses have account for major crops lost worldwide where more than 50% of their average yields were decreased yearly (Rodriguez et al. 2005). However, not all effects are detrimental. Plants are able to exhibit various molecular mechanisms as a defense system and these responses could be generally divided into three main groups. Firstly, signalling of stress-activated molecules leading to changes of osmotic and ionic homeostasis as well as detoxification mechanism. Secondly, up-regulation of different gene expression leading to synthesis of specific proteins (e.g. heat-shock proteins and LEA proteins) and some protective molecules (e.g. sugars, polyalcohols and amino acids). Thirdly, generation of reactive oxygen species (ROS) and activation of antioxidant systems by synthesizing secondary metabolites such as flavonoids and phenolic compounds (Boscaiu et al. 2008). Among these changes, synthesis of secondary metabolites is at the highest interest because it has a wide range of functions, ranging from plant defense against abiotic stresses to human benefits. Plants have the ability to produce vast variety of secondary metabolites naturally. Secondary metabolites have been defined as compounds that did not play a vital role in