Rozi Mohamed

Populus CEN/TFL1 regulates first onset of flowering, axillary meristem identity and dormancy release in Populus

Members of the CENTRORADIALIS (CEN)/TERMINAL FLOWER 1 (TFL1) subfamily control shoot meristem identity, and loss-of-function mutations in both monopodial and sympodial herbaceous plants result in dramatic changes in plant architecture. We studied the degree of conservation between herbaceous and woody perennial plants in shoot system regulation by overexpression and RNA interference (RNAi)-mediated suppression of poplar orthologs of CEN, and the related gene MOTHER OF FT AND TFL 1 (MFT). Field study of transgenic poplars (Populus spp.) for over 6 years showed that downregulation of PopCEN1 and its close paralog, PopCEN2, accelerated the onset of mature tree characteristics, including age of first flowering, number of inflorescences and proportion of short shoots. Surprisingly, terminal vegetative meristems remained indeterminate in PopCEN1-RNAi trees, suggesting the possibility that florigen signals are transported to axillary mersitems rather than the shoot apex. However, the axillary inflorescences (catkins) of PopCEN1-RNAi trees contained fewer flowers than did wild-type catkins, suggesting a possible role in maintaining the indeterminacy of the inflorescence apex. Expression of PopCEN1 was significantly correlated with delayed spring bud flush in multiple years, and in controlled environment experiments, 35S::PopCEN1 and RNAi transgenics required different chilling times to release dormancy. Considered together, these results indicate that PopCEN1/PopCEN2 help to integrate shoot developmental transitions that recur during each seasonal cycle with the age-related changes that occur over years of growth.

Genetic containment of forest plantations

Dispersal of pollen, seeds, or vegetative propagules from intensively bred, exotic, or recombinant DNA modified forest plantations may cause detrimental or beneficial ecological impacts on wild or managed ecosystems. Insertion of genes designed to prevent or substantially reduce dispersal could reduce the risk and extent of undesired impacts. Containment measures may also be required by law or marketplace constraints, regardless of risks or benefits. We discuss: (1) the context for when genetic containment or mitigation systems may be needed; (2) technology approaches and mechanisms; (3) the state of knowledge on genes/genomics of sexual reproduction in forest trees; (4) stability of transgene expression during vegetative growth; (5) simulation studies to define the level of containment needed; and (6) needed research to deliver effective containment technologies. We illustrate progress with several examples from our research on recombinant DNA modified poplars. Our simulations show that even partial sterility can provide very substantial reductions in gene flow into wild trees. We conclude that it is impossible to define the most effective containment approaches, nor their reliability, based on current genomic knowledge and technological tools. Additional genomic and technological studies of a wide variety of options are needed. Studies in field environments are essential to provide data relevant to ecological analysis and regulatory decisions and need to be carried out in phylogenetically diverse representatives of the economically most important taxa of forest trees.

Fungal diversity in wounded stems of Aquilaria malaccensis

Aquilaria malaccensis is a tropical tree which produces agarwood in its trunk often after being wounded and attacked by pathogens or insects. Fungi are generally viewed as the main microbial component responsible for agarwood formation. In this study, isolation of fungi from agarwood in damaged trees was carried out. Culture morphology and microscopic characteristics plus PCR amplification of the internal transcribed spacer (ITS) region from the fungal isolates as well as wood samples, were used to identify the fungal community composition of wounded A. malaccensis trees from a natural forest in West Malaysia. Conventional culture methodology revealed Cunninghamella, Curvularia, Fusarium and Trichoderma species as members of the agarwood community. Analysis of genomic DNA confirmed the identifications. When wood samples were used directly in PCR, an additional Lasiodiplodia species was identified. Neighbor-joining trees were constructed to examine the relationships between the isolates sequence data and reference sequences in GenBank. Five distinct clades resulted, supported with high bootstrap values, indicating the presence of five distinct taxa. The wounded trunks of A. malaccensis in the natural environment harbor multiple fungal taxa that exist in a complex system as a whole or in succession leading to agarwood production in the tree trunk.

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.

Cadmium toxicity induced alterations in the root proteome of green gram in contrasting response towards iron supplement

Cadmium signifies a severe threat to crop productivity and green gram is a notably iron sensitive plant which shows considerable variation towards cadmium stress. A gel-based proteomics analysis was performed with the roots of green gram exposed to iron and cadmium combined treatments. The resulting data show that twenty three proteins were down-regulated in iron-deprived roots either in the absence (−Fe/−Cd) or presence (−Fe/+Cd) of cadmium. These down-regulated proteins were however well expressed in roots under iron sufficient conditions, even in the presence of cadmium (+Fe/+Cd). The functional classification of these proteins determined that 21% of the proteins are associated with nutrient metabolism. The other proteins in higher quantities are involved in either transcription or translation regulation, and the rest are involved in biosynthesis metabolism, antioxidant pathways, molecular chaperones and stress response. On the other hand, several protein spots were also absent in roots in response to iron deprivation either in absence (−Fe/−Cd) or presence (−Fe/+Cd) of cadmium but were well expressed in the presence of iron (+Fe/+Cd). Results suggest that green gram plants exposed to cadmium stress are able to change the nutrient metabolic balance in roots, but in the mean time regulate cadmium toxicity through iron supplements.

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.

Effects of plant growth regulators, carbon sources and pH values on callus induction in Aquilaria malaccensis leaf explants and characteristics of the resultant calli

The endangered tropical tree, Aquilaria malaccensis, produces agarwood for use in fragrance and medicines. Efforts are currently underway to produce valuable agarwood compoundsn tissue culture. The purpose of this study was to develop an optimal growth medium, specifically, the best hormone combination for callus suspension culture. Using nursery-grown A. malaccensis, sterilized leaf explants were first incubated on basic Murashige and Skoog (MS) gel medium containing 15g/L sucrose and at pH 5.7. Different auxin types including 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-butyric acid (IBA), were tested at various concentrations (0.55, 1.1 and 1.65 μM) using the basic medium. Leaf explants were incubated for 30 days in the dark. Callus induced by 1.1 μM NAA had the highest biomass dry weight (DW) of 17.3 mg; however the callus was of a compact type. This auxin concentration was then combined with either 6-benzylaminopurine (BAP) or kinetin at 0.55, 1.1, 2.2 or 3.3 μM to induce growth of friable callus. The 1.1μM NAA + 2.2μM BAP combination produced friable callus with the highest biomass (93.3mg DW). When testing the different carbon sources and pHs, sucrose at 15g/L and pH at 5.7 yielded highest biomasses at 87.7mg and 83mg DW, respectively. Microscopic observations revealed the arrangement of the friable cells as loosely packed with relatively large cells, while for the compact callus, the cells were small and densely packed. We concluded that MS medium containing 15 g/L sucrose, 1.1 μM NAA + 2.2 μM BAP hormone combination, and a pH of 5.7 was highly effective for inducing friable callus from leaf explants of A. malaccensis for the purpose of establishing cell suspension culture.

DNA Barcoding of the Endangered Aquilaria (Thymelaeaceae) and Its Application in Species Authentication of Agarwood Products Traded in the Market.

The identification of Aquilaria species from their resinous non-wood product, the agarwood, is challenging as conventional techniques alone are unable to ascertain the species origin. Aquilaria is a highly protected species due to the excessive exploitation of its precious agarwood. Here, we applied the DNA barcoding technique to generate barcode sequences for Aquilaria species and later applied the barcodes to identify the source species of agarwood found in the market. We developed a reference DNA barcode library using eight candidate barcode loci (matK, rbcL, rpoB, rpoC1, psbA-trnH, trnL-trnF, ITS, and ITS2) amplified from 24 leaf accessions of seven Aquilaria species obtained from living trees. Our results indicated that all single barcodes can be easily amplified and sequenced with the selected primers. The combination of trnL-trnF+ITS and trnL-trnF+ITS2 yielded the greatest species resolution using the least number of loci combination, while matK+trnL-trnF+ITS showed potential in detecting the geographical origins of Aquilaria species. We propose trnL-trnF+ITS2 as the best candidate barcode for Aquilaria as ITS2 has a shorter sequence length compared to ITS, which eases PCR amplification especially when using degraded DNA samples such as those extracted from processed agarwood products. A blind test conducted on eight agarwood samples in different forms using the proposed barcode combination proved successful in their identification up to the species level. Such potential of DNA barcoding in identifying the source species of agarwood will contribute to the international timber trade control, by providing an effective method for species identification and product authentication.

Understanding Agarwood Formation and Its Challenges

The resinous portion of the Aquilaria tree is called agarwood, a valuable non-timber product being used as medicine and incenses in Asia, Middle East, and Europe. Driven by high demand, the wild resources of agarwood-producing trees have been greatly threatened. This fragrant product contains many aromatic substances and is obtained from the pathological conditions of the wood of living trees. The knowledge regarding the technology for inducing agarwood and its continuous formation in the tree is still limited. To conserve the wild Aquilaria spp. and to supply sustainable amount of agarwood, cultivation of Aquilaria trees in combination with induction through artificial technique is seen as the best approach. In this chapter we will discuss the fundamentals of agarwood formation in the producing trees, the molecular pathway in its synthesis, current methods applied for agarwood induction in cultivated trees, and finally the factors influencing agarwood yield and quality.

The Origin and Domestication of Aquilaria, an Important Agarwood-Producing Genus

The Aquilaria (Thymelaeaceae) tree is a well-known important agarwood-producing genus, which is endemic to the Indomalesia region. The genus is currently protected under CITES regulation and the IUCN Red List due to its heavy declination in the natural population in various sourcing countries. Derived from its precious non-wood fragrant products, the genus was given different names throughout the history until it was finalized in 1783. To date, there are 21 recognized Aquilaria species recorded, of which 13 are reportedly fragrant resin producers, and the status of the remaining eight Aquilaria species is yet to be investigated. Aquilaria is heavily exploited in the wild due to the destructive agarwood harvesting technique that requires hacking of the wood parts to induce agarwood production. Various conservation efforts have been carried out to avoid further destruction toward its gene pool. This includes introducing the species for cultivation and planting the trees in large plantations or home gardens, which further provide a sustainable agarwood production in the industry and indirectly contribute to the local economy. At present, an accurate classification of Aquilaria species is yet to be achieved; misidentification happens frequently, either genuinely because of lack of information and training or intentionally for business gains. In conclusion, a proper taxonomy and classification system are essential for conserving Aquilaria species genetic diversity and for identifying species origin of agarwood products aimed at international trade control.