KEY MESSAGE: The oil palm EgPAL1 gene promoter and its regulatory region were functional as a promoter in the heterologous system of Arabidopsis according to the cis-acting elements present in that region. The promoter was developmentally regulated, vascular tissue specific and responsive to water stress agents. Phenylalanine ammonia lyase (PAL, EC 220.127.116.11) is the key enzyme of the phenylpropanoid pathway which plays important roles in plant development and adaptation. To date, there is no report on the study of PAL from oil palm (Elaeis guineensis), an economically important oil crop. In this study, the 5' regulatory sequence of a highly divergent oil palm PAL gene (EgPAL1) was isolated and fused with GUS in Arabidopsis to create two transgenic plants carrying the minimal promoter with (2302 bp) and without its regulatory elements (139 bp). The regulatory sequence contained cis-acting elements known to be important for plant development and stress response including the AC-II element for lignin biosynthesis and several stress responsive elements. The promoter and its regulatory region were fully functional in Arabidopsis. Its activities were characterised by two common fundamental features of PAL which are responsive to plant internal developmental programme and external factors. The promoter was developmentally regulated in certain organs; highly active in young organs but less active or inactive in mature organs. The presence of the AC elements and global activity of the EgPAL1 promoter in all organs resembled the property of lignin-related genes. The existence of the MBS element and enhancement of the promoter activity by PEG reflected the behaviour of drought-responsive genes. Our findings provide a platform for evaluating oil palm gene promoters in the heterologous system of Arabidopsis and give insights into the activities of EgPAL1 promoter in oil palm.
KEY MESSAGE: TAAAAT and a novel motif, GCTTCA found in the oil palm stearoyl-ACP desaturase (SAD1) promoter are involved in regulating mesocarp-specific expression. Two key fatty acid biosynthetic genes, stearoyl-ACP desaturase (SAD1), and acyl-carrier protein (ACP3) in Elaeis guineensis (oil palm) showed high level of expression during the period of oil synthesis in the mesocarp [12-19 weeks after anthesis (w.a.a.)] and kernel (12-15 w.a.a.). Both genes are expressed in spear leaves at much lower levels and the expression increased by 1.5-fold to 2.5-fold following treatments with ethylene and abscisic acid (ABA). Both SAD1 and ACP3 promoters contain phytohormone-responsive, light-responsive, abiotic factors/wounding-responsive, endosperm specificity and fruit maturation/ripening regulatory motifs. The activities of the full length and six 5' deletion fragments of the SAD1 promoter were analyzed in transiently transformed oil palm tissues by quantitative β-glucuronidase (GUS) fluorometric assay. The highest SAD1 promoter activity was observed in the mesocarp followed by kernel and the least in the leaves. GUS activity in the D3 deletion construct (- 486 to + 108) was the highest, while the D2 (- 535 to + 108) gave the lowest suggesting the presence of negative cis-acting regulatory element(s) in the deleted - 535 to - 486 (49 bp). It was found that the 49-bp region binds to the nuclear protein extract from mesocarp but not from leaves in electrophoretic mobility shift assay (EMSA). Further fine-tuned analysis of this 49-bp region using truncated DNA led to the identification of GCTTCA as a novel motif in the SAD1 promoter. Interestingly, another known fruit ripening-related motif, LECPLEACS2 (TAAAAT) was found to be required for effective binding of the novel motif to the mesocarp nuclear protein extract.
The efficiency of GUS (β-Glucuronidase) gene expression in embryogenic callus and young leaflets of mature and seedling palm after microprojectile bombardment with five constructs (pEmuGN, pAHC25, pAct1-F4, pGH24 and pBARGUS) was evaluated to identify the most suitable promoter(s) to use in transformation attempts in oil palm. Expression of the GUS gene driven by theEmu, Ubi1, Act1 35S orAdh1 was assayed, both histochemically and fluorometrically, from a total of 200 plates of tissues in eight independent experiments two days after bombardment. A completely randomized experimental design was used for each experiment, and the data analysed by ANOVA and Duncan's Multiple Range Test. The expression level of GUS driven by theEmu orUbi1 promoters was significantly higher than that of the Act], 35S and Adhl promoters in many experiments, and that of theAdhl was significantly lower than those of the other four promoters. Both histochemical and fluorometric data indicate that in embryogenic callus, the expression of theEmu promoter was higher than that of theUbi1 whereas in young leaflets from mature palm the Ubi1 expression was stronger. The performances of the five promoters were also tested in tobacco callus using a fluorometric GUS assay. The activity of the 35S promoter was highest, and significantly different from that of all the other promoters except theEmu, and that of theAct1 promoter was lowest. These results indicate that either theUbil orEmu promoter should facilitate the expression of desired genes in oil palm and aid in development of an efficient stable transformation system.
Hevea brasiliensis anther calli were genetically transformed using Agrobacterium GV2260 (p35SGUSINT) that harboured the β-glucuronidase (gus) and neomycin phosphotransferase (nptII) genes. β-Glucuronidase protein (GUS) was expressed in the leaves of kanamycin-resistant plants that were regnerated, and the presence of the gene was confirmed by Southern analysis. GUS was also observed to be expressed in the latex and more importantly in the serum fraction. Transverse sections of the leaf petiole from a transformed plant revealed GUS expression to be especially enhanced in the phloem and laticifers. GUS expression was subsequently detected in every one of 194 plants representing three successive vegetative cycles propagated from the original transformant. Transgenic Hevea could thus facilitate the continual production of foreign proteins expressed in the latex.
The technique of mRNA fingerprinting was used to isolate flower-specific cDNAs in the oil palm. Differences in the RNA populations between vegetative tissue (leaf) and inflorescences at various stages of flower development were examined using 18 primer combinations. A total of 16 flower-specific cDNAs were identified, of which 15 were successfully re-amplified. Reverse Northern analysis confirmed that 8 of the 15 cDNAs appeared to truly represent differentially expressed mRNAs in flowering tissues. Northern blot analysis subsequently showed that 5 of the clones are preferentially or exclusively expressed in the flowering tissues of oil palm.
Plant cells are characterized by a unique group of interconvertible organelles called plastids, which are descended from prokaryotic endosymbionts. The most studied plastid type is the chloroplast, which carries out the ancestral plastid function of photosynthesis. During the course of evolution, plastid activities were increasingly integrated with cellular metabolism and functions, and plant developmental processes, and this led to the creation of new types of non-photosynthetic plastids. These include the chromoplast, a carotenoid-rich organelle typically found in flowers and fruits. Here, we provide an introduction to non-photosynthetic plastids, and then review the structures and functions of chromoplasts in detail. The role of chromoplast differentiation in fruit ripening in particular is explored, and the factors that govern plastid development are examined, including hormonal regulation, gene expression, and plastid protein import. In the latter process, nucleus-encoded preproteins must pass through two successive protein translocons in the outer and inner envelope membranes of the plastid; these are known as TOC and TIC (translocon at the outer/inner chloroplast envelope), respectively. The discovery of SP1 (suppressor of ppi1 locus1), which encodes a RING-type ubiquitin E3 ligase localized in the plastid outer envelope membrane, revealed that plastid protein import is regulated through the selective targeting of TOC complexes for degradation by the ubiquitin-proteasome system. This suggests the possibility of engineering plastid protein import in novel crop improvement strategies.
Recent developments in modern biotechnology such as the use of RNA interference (RNAi) have broadened the scope of crop genetic modification. RNAi strategies have led to significant achievements in crop protection against biotic and abiotic stresses, modification of plant traits, and yield improvement. As RNAi-derived varieties of crops become more useful in the field, it is important to examine the capacity of current regulatory systems to deal with such varieties, and to determine if changes are needed to improve the existing frameworks. We review the biosafety frameworks from the perspective of developing countries that are increasingly involved in modern biotechnology research, including RNAi applications, and make some recommendations. Malaysia and India have approved laws regulating living modified organisms and products thereof, highlighting that the use of any genetically modified step requires regulatory scrutiny. In view of production methods for exogenously applied double-stranded RNAs and potential risks from the resulting double-stranded RNA-based products, we argue that a process-based system may be inappropriate for the non-transformative RNAi technology. We here propose that the current legislation needs rewording to take account of the non-transgenic RNAi technology, and discuss the best alternative for regulatory systems in India and Malaysia in comparison with the existing frameworks in other countries.
KEY MESSAGE: Three tau class MaGSTs responded to abiotic stress, MaGSTF1 and MaGSTL1 responded to signaling molecules, they may play an important role in the growth of banana plantlet. Glutathione S-transferases (GST) are multifunctional detoxification enzymes that participate in a variety of cellular processes, including stress responses. In this study, we report the molecular characteristics of five GST genes (MaGSTU1, MaGSTU2, MaGSTU3, MaGSTF1 and MaGSTL1) cloned from banana (Musa acuminate L. AAA group, cv. Cavendish) using a RACE-PCR-based strategy. The predicted molecular masses of these GSTs range from 23.4 to 27.7 kDa and their pIs are acidic. At the amino acid level, they share high sequence similarity with GSTs in the banana DH-Pahang (AA group) genome. Phylogenetic analysis showed that the deduced amino acid sequences of MaGSTs also have high similarity to GSTs of other plant species. Expression analysis by semi-quantitative RT-PCR revealed that these genes are differentially expressed in various tissues. In addition, their expression is regulated by various stress conditions, including exposure to signaling molecules, cold, salinity, drought and Fusarium oxysporum f specialis(f. Sp) cubense Tropical Race 4 (Foc TR4) infection. The expression of the tau class MaGSTs (MaGSTU1, MaGSTU2 and MaGSTU3) mainly responded to cold, salinity and drought while MaGSTF1 and MaGSTL1 expressions were upregulated by signaling molecules. Our findings suggest that MaGSTs play a key role in both development and abiotic stress responses.
Oil palm is a major economic crop for Malaysia. The major challenges faced by the industry are labor shortage, availability of arable land and unstable commodity price. This has caused the industry to diversify its applications into higher value products besides increasing its yield. While conventional breeding has its limitations, biotechnology was identified as one of the tools for overcoming the above challenges. Research on biotechnology of oil palm began more than two decades ago leveraging a multidisciplinary approach involving biochemical studies, gene and promoter isolation, transformation vector construction and finally genetic transformation to produce the targeted products. The main target of oil palm biotechnology research is to increase oleic acid in the mesocarp. Other targets are stearic acid, palmitoleic acid, ricinoleic acid, lycopene (carotenoid) and biodegradable plastics. Significant achievements were reported for the biochemical studies, isolation of useful oil palm genes and characterization of important promoters. A large number of transformation constructs for various targeted products were successfully produced using the isolated oil palm genes and promoters. Finally transformation of these constructs into oil palm embryogenic calli was carried out while the regeneration of transgenic oil palm harboring the useful genes is in progress.
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.
The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms.
The Brassica napus secondary embryogenesis system requires no exogenous growth regulator to stimulate embryo development. It is stable embryogenically over a long period of culture and has a distinct pre-embryogenic stage. This system was used to investigate the morphological and cellular changes occurring in the embryogenic tissue compared to non-embryogenic tissue using various microscopy techniques. A unique ultrastructural feature designated the extracellular matrix (ECM) was observed on the surface of pre-embryogenic embryoids but not on the non-embryogenic individuals. The ECM layer was found to be dominant in the pre-embryogenic stage and reduced to fragments during embryo growth and development in mature embryogenic tissue. This is a novel aspect of the phenotype previously unreported in the Brassica system. This structure might be linked to acquisition of embryogenic competence.
A natural shift is taking place in the approaches being adopted by plant scientists in response to the accessibility of systems-based technology platforms. Metabolomics is one such field, which involves a comprehensive non-biased analysis of metabolites in a given cell at a specific time. This review briefly introduces the emerging field and a range of analytical techniques that are most useful in metabolomics when combined with computational approaches in data analyses. Using cases from Arabidopsis and other selected plant systems, this review highlights how information can be integrated from metabolomics and other functional genomics platforms to obtain a global picture of plant cellular responses. We discuss how metabolomics is enabling large-scale and parallel interrogation of cell states under different stages of development and defined environmental conditions to uncover novel interactions among various pathways. Finally, we discuss selected applications of metabolomics.
The effects of medium strategies [maintenance (M), intermediary (G), and production (P) medium] on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H2O2) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated. These were compared with third-stage leaf and 1-month-old callus culture. With P medium strategy, cell growth at 49 g l(-1), intracellular AQ content at 42 mg g(-1) DW, and H2O2 level at 9 micromol g(-1) FW medium were the highest as compared to the others. However, the extent of lipid peroxidation at 40.4 nmol g(-1) FW and total carotenoids at 13.3 mg g(-1) FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H2O2 levels. Vitamin C content at 30-120 microg g(-1) FW in all culture systems was almost half the leaf content. On the other hand, vitamin E content was around 400-500 microg g(-1) FW in 7-day-old cultures from all medium strategies and reduced to 50-150 microg g(-1) FW on day 14 and 21; as compared to 60 microg g(-1) FW in callus and 200 microg g(-1) FW in the leaf. This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures. When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants.
Three different morphological callus types, identified as type A, B and C, and tips of in vitro inflorescences were used as target tissues for genetic transformation. Five different DNA plasmids carrying a synthetic green fluorescent protein (gfp) gene driven by different promoters, CaMV 35S, HBT, and Ubi1 were tested for the genetic transformation of Dendrobium Sonia 17. 35S-sgfp-TYG-nos (p35S) with the CaMV 35S promoter showed the highest GFP transient expression rate, while the HBT and Ubi1 promoters showed a relatively lower expression rate in all of the target tissues tested. The highest number of GFP-expressing cells was observed on day 2 post-bombardment, and the number declined gradually over the course of the next 2 weeks. Type A and B callus were found to be the best potential target tissues for genetic transformation.
Regeneration of plantlets from shoot apex-derived callus and "calloid" cultures of a local taro [Colocasia esculenta var. antiquorum cv. Keladi Birah] cultivar, was expedited by treatment with high levels of spermine. The total time taken, from culture of primary shoot apices on modified Linsmaier and Skoog medium supplemented with trichlorophenoxyacetic acid and kinetin, to complete plantlet regeneration, was reduced by 2-16 weeks, when the callus and "calloid" cultures were treated with 0.01, 0.1 and 1 mM spermine. Furthermore, the number of plantlets produced per gram callus increased from 25 to 55. On media supplemented with arginine and ornithine, no callus was initiated from expiants and no plantlets differentiated from pre-established callus.
In vitro growth and multiplication of taro [Colocasia esculenta var. antiquorum cv. Keladi Birah] was improved considerably, when primary shoot apices were cultured on two modifications of Linsmaier and Skoog  medium, containing 5.5 mg 1(-1) naphthaleneacetic acid and 0.2 mg 1(-1) kinetin or 1.85 mg 1(-1) naphthaleneacetic acid and 2 mg 1(-1) kinetin and supplemented with 10(-4) or 10(-3) mol·1(-1) of polyamine spermine or either of the precursors of polyamine putrescine-arginine and ornithine. Plantlets were regenerated directly from primary shoot apices, axillary buds and protocorm-like bodies [PLB]. Frequency of plantlet regeneration, rate of development and growth in height of main plantlets were enhanced by the addition of arginine and ornithine to the media. Secondary plantlet formation from axillary buds and PLB were promoted by spermine and arginine respectively.
KEY MESSAGE: This study highlights the changes in umami-related nucleotide and glutamate levels when the AMP deaminase gene was elevated in transgenic tomato. Taste is perceived as one of a combination of five sensations, sweet, sour, bitter, salty, and umami. The umami taste is best known as a savoury sensation and plays a central role in food flavour, palatability, and eating satisfaction. Umami flavour can be imparted by the presence of glutamate and is greatly enhanced by the addition of ribonucleotides, such as inosine monophosphate (IMP) and guanosine monophosphate (GMP). The production of IMP is regulated by the enzyme adenosine monophosphate (AMP) deaminase which functions to convert AMP into IMP. We have generated transgenic tomato (Solanum lycopersicum) lines over expressing AMP deaminase under the control of a fruit-specific promoter. The transgenic lines showed substantially enhanced levels of AMP deaminase expression in comparison to the wild-type control. Elevated AMP deaminase levels resulted in the reduced accumulation of glutamate and increased levels of the umami nucleotide GMP. AMP concentrations were unchanged. The effects on the levels of glutamate and GMP were unexpected and are discussed in relation to the metabolite flux within this pathway.
KEY MESSAGE: Several hypomethylated sites within the Karma region of EgDEF1 and hotspot regions in chromosomes 1, 2, 3, and 5 may be associated with mantling. One of the main challenges faced by the oil palm industry is fruit abnormalities, such as the "mantled" phenotype that can lead to reduced yields. This clonal abnormality is an epigenetic phenomenon and has been linked to the hypomethylation of a transposable element within the EgDEF1 gene. To understand the epigenome changes in clones, methylomes of clonal oil palms were compared to methylomes of seedling-derived oil palms. Whole-genome bisulfite sequencing data from seedlings, normal, and mantled clones were analyzed to determine and compare the context-specific DNA methylomes. In seedlings, coding and regulatory regions are generally hypomethylated while introns and repeats are extensively methylated. Genes with a low number of guanines and cytosines in the third position of codons (GC3-poor genes) were increasingly methylated towards their 3' region, while GC3-rich genes remain demethylated, similar to patterns in other eukaryotic species. Predicted promoter regions were generally hypomethylated in seedlings. In clones, CG, CHG, and CHH methylation levels generally decreased in functionally important regions, such as promoters, 5' UTRs, and coding regions. Although random regions were found to be hypomethylated in clonal genomes, hypomethylation of certain hotspot regions may be associated with the clonal mantling phenotype. Our findings, therefore, suggest other hypomethylated CHG sites within the Karma of EgDEF1 and hypomethylated hotspot regions in chromosomes 1, 2, 3 and 5, are associated with mantling.
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.