Crude extract of Trichoderma elicits agarwood substances in cell suspensionculture of the tropical tree, Aquilaria malaccensis Lam.

Agarwood is the precious fragrant wood produced by the tropical tree Aquilaria, often after elicitation by wounding or fungal attack. In this study we established a cell suspension culture of A. malaccensis from leaf-derived callus and induced agarwood production in the culture using fungal elicitors. Elicitors were made from crude mycelial extracts of two fungal species from the genera Trichoderma and Lasidiplodia. The elicitors were added to the cell suspension culture, initiated with 2 g of fresh calli, at concentrations ranging from 2 to 10 mg L-1. A light agarwood scent was detected from the suspension culture elicited with 8 mg L-1 Trichoderma extract. To increase scent intensity, cell suspension cultures were initiated from 2 to 8 g of calli and treated with 8 mg L-1 Trichoderma extract. The combination of 8 g of calli inoculum and 8 mg L-1 Trichoderma extract produced the most intense fragrance, one comparable to agarwood scent. The cell culture was harvested, extracted in methanol, and analyzed using GC-MS. Several important agarwood compounds were detected including 8-epi-.gama.-eudesmol, á-guaiene, and alloaromadendrene oxide-1. Trichoderma appeared to be a suitable inducer for agarwood production when used at an optimal concentration and in combination with a cell suspension culture of Aquilaria

Plant cytochrome P450s: nomenclature and involvement in natural product biosynthesis

Cytochrome P450s constitute the largest family of enzymatic proteins in plants acting on various endogenous and xenobiotic molecules. They are monooxygenases that insert one oxygen atom into inert hydrophobic molecules to make them more reactive and hydro-soluble. Besides for physiological functions, the extremely versatile cytochrome P450 biocatalysts are highly demanded in the fields of biotechnology, medicine, and phytoremediation. The nature of reactions catalyzed by P450s is irreversible, which makes these enzymes attractions in the evolution of plant metabolic pathways. P450s are prime targets in metabolic engineering approaches for improving plant defense against insects and pathogens and for production of secondary metabolites such as the anti-neoplastic drugs taxol or indole alkaloids. The emerging examples of P450 involvement in natural product synthesis in traditional medicinal plant species are becoming increasingly interesting, as they provide new alternatives to modern medicines. In view of the divergent roles of P450s, we review their classification and nomenclature, functions and evolution, role in biosynthesis of secondary metabolites, and use as tools in pharmacology

Cloning of Phenylalanine ammonia-lyase (PAL) gene fragment from Aquilaria malaccensis Lam. (karas).

Current practice of indiscriminate and illegal felling of 'karas' trees (Aquilaria malaccensis) to extract 'gaharu' in the wild have caused the population to dwindle drastically. 'Gaharu' formed as a result of natural defense mechanism in trees, which unfortunately happens over a long period of time. To better understand natural processes of gaharu-making, we sought to study the roles of defense genes in 'karas' by cloning a gene that synthesizes secondary compounds. The phenylalanine ammonia-lyase (PAL) gene encodes an enzyme in the metabolism of phenylpropanoid compounds, which are produced in response to stress, such as damage by wounding and pathogen attack. We report a cloned PAL gene fragment from A. malaccensis using the polymerase chain reaction (PCR) technique. The fragment was amplified from genomic DNA of a wounded 2-year old tree and cloned into the pGEM-T Easy Vector. The cloned fragment had a length of 610bp and an uninterrupted open reading frame of 201 amino acids. When searched against the Genebank database using BLAST tools, the cloned fragment shared 92% sequence similarity at amino acid level to known PAL proteins. This work will serve as a foundation in identifying important genes for triggering 'gaharu' synthesis

Rediscovery of Aquilaria rostrata (Thymelaeaceae), a species thought to be extinct, and notes on Aquilaria conservation in Peninsular Malaysia

After more than 100 years since the first discovery, Aquilaria rostrata, a critically endangered species listed in the IUCN Red List and presumably extinct, has been rediscovered in Terengganu State of Peninsular Malaysia. Here, we describe the history, taxonomy, ecology and conservation status of this endemic species, and compare our findings with the species description made from the first and only collection produced prior to this study. In addition, we present the similarities between A. rostrata and several Aquilaria species occurring in Peninsular Malaysia and neighbouring regions using molecular sequence data from the nuclear ribosomal DNA (Internal Transcribed Spacer) and chloroplast intergenic spacer region (trnL-trnF). Our morphological and sequence analyses support the separate status of A. rostrata, a long-lost endemic species of Malaysia

Microscopic observation of `Gaharu` wood from Aquilaria malaccensis

Aquilaria produces fragrant wood known as `Gaharu` in its stem and branches, often in mature and damaged trees. In this study, anatomical characteristics in juvenile and mature trees were investigated by comparing their anatomical structures after various staining methods and direct observations under a light microscope. Juvenile and mature wood share similar anatomical structures. No major differences were observed other than the percentage of area covered by included phloem in juvenile was 2.16 times more than that of the mature wood. Microscopic observation revealed that in mature resinous wood, brownish bodies were found in ray and axial parenchyma, included phloem, xylem vessels and fibres, and this finding indicates that these are important elements for `Gaharu` depositing. Thus, it was concluded that juvenile tree possess the anatomical features of that of mature wood in producing `Gaharu`

Comparisons of different RNA extraction methods on woody tissues of the tropical tree, Aquilaria malaccensis

Aquilaria malaccensis (karas) produces oleoresin in its wood (known as agarwood) when responding to wounding and microbial infection. Wood tissues are known to contain high levels of polysaccharides, polyphenolics and secondary metabolites, which make RNA extraction challenging. In this work, six different methods for extracting RNA from wood tissues of A. malaccensis were compared. RNA yield, purity, and integrity number, were used as parameters to evaluate the efficiency of each method. Conventional methods yielded RNA with good purity but the RNA integrity was poor. The commercial RNeasy Plant Mini kit protocol was modified by means of scaling-up the reaction and combining all aliquots in the same RNeasy spin column, and yielded the highest yield while maintaining the integrity of the RNA. We found that this kit with some modifications was most suitable for extracting RNA from healthy wood and agarwood. This study is essential for future molecular studies on agarwood

Chemical characterization of imperata cylindrical (`Lalang`) and pennisetum purpureum (Napier grass) for bioethanol production in Malaysia

Grass is a candidate biomass producer because it is fibrous and it thrives well on poor soils. The chemical properties of two grass species growing naturally and abundantly on idle lands in Malaysia were investigated in this study. The grasses selected were Imperata cylindrical (`Lalang`) and Pennisetum purpureum (Napier grass). For the purpose of analysis, Napier grass was further divided into male and female plants, and stem and leaves. Lignin, hemicellulose and cellulose contents were determined using the TAPPI standard methods. `Lalang` was found not to be an attractive biomass producer because of its high lignin content (22%) (P<0.05). On the contrary, Napier grass, particularly the female stem, had low lignin content (13%) (P<0.05) and a favourably high level of cellulose (46%) (P<0.05). In the female leaf, lignin content was higher (20.7%), while the cellulose content (30.4%) was lower compared to the stem. Although the cellulose content in the male stem (51%) was slightly higher (P<0.05) than the female, its lignin was two-fold above that of the female stem, making it a less desirable biomass producer. Hence, it was concluded that female Napier grass has a good potential of becoming a biomass producer in bioethanol production in Malaysia

Genetic engineering of disease resistance in poplar : effects of bacterio-opsin over-expression and analysis of a copper-based system for resistance-gene activation

We tested a synthetic bacterio-opsin (bO) gene derived from Halobacterium halobium for its effect on disease resistance in transgenic poplar hybrids, and assessed a genetic system for controlling expression of this gene. In tobacco the bO gene caused necrotic lesions similar to an induced hypersensitive response (HR), increased levels of several defense-related mRINAs, and enhanced resistance to pathogens (Mittler et al. 1995). Our goal was to test whether the bO gene could also promote the HR response and induce resistance against pathogens of poplar. Using Agrobacterium transformation, we produced 35 transgenic lines within three hybrid poplar clones. Expression of the bO gene was under the control of the cauliflower mosaic virus (CaMV) 35S promoter and ) translational enhancer, and the protein included a transit peptide to target it to the chloroplast. Transgenic lines were confirmed through their ability to root on kanamycincontaining medium, PCR amplification using bO-specific primers, and RNA blots probed with the bO gene. Necrotic lesions induced by the bO gene were seen predominantly on the lower and older leaves of greenhouse- and field-grown trees, similar to that reported in tobacco. Most lines displayed low to moderate numbers of lesions (mean lesion index=6.6, coefficient of variation=106%); however, one line had a very high lesion index (6-fold greater than the mean). Based on northern blots, bO expression among lines also varied widely (coefficient of variation=90%). We studied the effect of the bO gene on the expression of two types of defense-related genes, phenylalanine ammonia-lyase (PAL]), and two wound-inducible (win) chitinases, win6 and win8. Because of the low constitutive expression of the chitinases in poplar, their expression was induced by mechanical wounding prior to RINA extraction. No significant change in expression was found for any of these genes associated with the expression of the bO gene, including the line with very high bO expression. Resistance to different fungal pathogens was examined in the field and greenhouse after artificial inoculation. When field-grown trees were inoculated with fungal spores of Venturia leaf and shoot blight, bO-containing transgenics did not differ significantly from non-transgenic plants of the same clone (195-529). When clone 189-434, which is susceptible to the rust pathogen Melampsora occidentalis, was inoculated in the greenhouse, transgenic and control plants were also not significantly different in disease development. When all the lines were tested for susceptibility to two Septoria leaf and stem pathogens using in vitro inoculation, transgenics and controls were again similar in susceptibility. It was concluded that even though the bO gene was over-expressed and induced an HRmimic, lesion phenotype in some lines, it did not induce broad-spectrum defenserelated genes, and it failed to improve fungal disease resistance. To test a system that might be suitable for regulating expression of disease resistance transgenes in poplar, we studied the copper-based gene activation system reported to be effective in tobacco. Using both histochemical and fluorometric assays with a GUS reporter gene, we found that transgenic poplar leaves with both control (promoter-GUS only) and induction (promoter-GUS plus the copper binding ACE] transcription factor) constructs displayed constitutive expression without the addition of exogenous copper (basal medium concentration was 0.032 uM CuSO4). With the full induction construct, expression varied in a complex manner as a function of copper concentration (0 - 100 uM). It was concluded that because of the presence of endogenous transcription factors or inducing ions that compete with copper, the system is unlikely to be useful for regulating gene expression in poplar leaves

Expression and Function of Populus Homologs to TERMINAL FLOWER 1 Genes: Roles in Onset of Flowering and Shoot Phenology

We isolated and characterized the expression of two genes from Populus trichocarpa that are homologous to the TERMINAL FLOWER 1 (TFL1) gene from the model annual plant Arabidopsis. In Arabidopsis, overexpression of the TFL1 gene extends the vegetative growth phase, and the homozygous mutant tfl1 allele causes early flowering and formation of a terminal flower. Overexpression of another TFL1 family member, FLOWERING LOCUS T (FT), inhibits the action of TFL1. The two homologs studied, poplar CENTRORADIALIS LIKE-1 (PtCENL-1) and poplar MOTHER OF FT AND TFL1 (PtMFT), encode proteins that are 52% identical to one another; and 72% and 50% identical to TFL1, respectively. Real-time RT-PCR studies revealed that PtCENL-1 was expressed in all stages of development studied, and was most strongly expressed in vegetative buds and shoot apices. PtMFT was expressed preferentially in inflorescence buds. Expression patterns suggest that PtCENL-1 promotes maintenance of the vegetative growth phase, and that PtMFT promotes the onset of flowering. We tested these hypotheses by overexpression of the PtCENL-1/PtMFT under the control of the CaMV 35S promoter (poplar and Arabidopsis), and via suppression of the endogenous genes via RNA interference (RNAi: poplar only). Some PtCENL-1 RNAi trees flowered during the second growing season in the field, several years earlier than expected. Floral buds were detected in four independent gene insertion events; in two of the four events, floral buds expanded into mature-appearing female catkins and dehisced, though seeds were not formed. All four events had native PtCENL-1 transcript levels lower than in non-flowering events, and below 50% of the level detected in non-transgenic poplar. These early flowering events had normal budflush, however, 35S::PtCENL-1 transcript levels were strongly and positively correlated with date of budflush. These results suggest that endogenous PtCENL-1 is a natural inhibitor of the onset of flowering, and may also retard release from vegetative dormancy. Correspondingly, ectopic expression of PtCENL-1 in Arabidopsis had delayed flowering. No phenotypic differences were observed in PtMFT overexpressing or RNAi transgenic trees. However, ectopic expression of PtMFT in Arabidopsis caused early flowering. Suppression of PtCENL-1 might be useful for inducing early flowering in Populus

Fungal inoculation induces agarwood in young Aquilaria malaccensis trees in the nursery

Fungi are often used to induce agarwood in Aquilaria trees. This study was conducted to evaluate the effect of several fungi on agarwood formation over time in young Aquilaria malaccensis (Lam.) trees. Typical changes in the length and light intensity of the resulting discoloration were observed after three and six month periods following inoculation. Wood samples were observed microscopically and classified into several light intensity groups. The discoloration length was measured longitudinally. The duration after inoculation affected the mean of discoloration length: the 6-month old sample (1.70 cm) had a wider discoloration zone when compared to the 3-month old sample (1.17 cm). When measuring the discoloration intensity, a positive relationship with time was perceived. Digital images, captured using a camera-equipped microscope, revealed that wood samples collected after six months appeared to be 1.8-times darker than after three months. We concluded that time, not the species of any of the tested fungi, had significant effect on discoloration length and intensity. Gas chromatography/mass spectrometry (GCMS) analysis of the 6-month old sample yielded some important agarwood compounds such as benzylacetone, anisylacetone, guaiene and palustrol. This demonstrates that the tested fungi have the ability to induce agarwood formation in nursery A. malaccensis trees