Aidyn Mouradov

Flavonoids: a metabolic network mediating plants adaptation to their real estate

From an evolutionary perspective, the emergence of the sophisticated chemical scaffolds of flavonoid molecules represents a key step in the colonization of Earth’s terrestrial environment by vascular plants nearly 500 million years ago. The subsequent evolution of flavonoids through recruitment and modification of ancestors involved in primary metabolism has allowed vascular plants to cope with pathogen invasion and damaging UV light. The functional properties of flavonoids as a unique combination of different classes of compounds vary significantly depending on the demands of their local real estate. Apart from geographical location, the composition of flavonoids is largely dependent on the plant species, their developmental stage, tissue type, subcellular localization, and key ecological influences of both biotic and abiotic origin. Molecular and metabolic cross-talk between flavonoid and other pathways as a result of the re-direction of intermediate molecules have been well investigated. This metabolic plasticity is a key factor in plant adaptive strength and is of paramount importance for early land plants adaptation to their local ecosystems. In human and animal health the biological and pharmacological activities of flavonoids have been investigated in great depth and have shown a wide range of anti-inflammatory, anti-oxidant, anti-microbial, and anti-cancer properties. In this paper we review the application of advanced gene technologies for targeted reprogramming of the flavonoid pathway in plants to understand its molecular functions and explore opportunities for major improvements in forage plants enhancing animal health and production.

Co-Cultivation of Fungal and Microalgal Cells as an Efficient System for Harvesting Microalgal Cells, Lipid Production and Wastewater Treatment

The challenges which the large scale microalgal industry is facing are associated with the high cost of key operations such as harvesting, nutrient supply and oil extraction. The high-energy input for harvesting makes current commercial microalgal biodiesel production economically unfeasible and can account for up to 50% of the total cost of biofuel production. Co-cultivation of fungal and microalgal cells is getting increasing attention because of high efficiency of bio-flocculation of microalgal cells with no requirement for added chemicals and low energy inputs. Moreover, some fungal and microalgal strains are well known for their exceptional ability to purify wastewater, generating biomass that represents a renewable and sustainable feedstock for biofuel production. We have screened the flocculation efficiency of the filamentous fungus A. fumigatus against 11 microalgae representing freshwater, marine, small (5 µm), large (over 300 µm), heterotrophic, photoautotrophic, motile and non-motile strains. Some of the strains are commercially used for biofuel production. Lipid production and composition were analysed in fungal-algal pellets grown on media containing alternative carbon, nitrogen and phosphorus sources contained in wheat straw and swine wastewater, respectively. Co-cultivation of algae and A. fumigatus cells showed additive and synergistic effects on biomass production, lipid yield and wastewater bioremediation efficiency. Analysis of fungal-algal pellets fatty acids composition suggested that it can be tailored and optimised through co-cultivating different algae and fungi without the need for genetic modification.

Fungal-assisted algal flocculation: application in wastewater treatment and biofuel production

BackgroundThe microalgal-based industries are facing a number of important challenges that in turn affect their economic viability. Arguably the most important of these are associated with the high costs of harvesting and dewatering of the microalgal cells, the costs and sustainability of nutrient supplies and costly methods for large scale oil extraction. Existing harvesting technologies, which can account for up to 50% of the total cost, are not economically feasible because of either requiring too much energy or the addition of chemicals. Fungal-assisted flocculation is currently receiving increased attention because of its high harvesting efficiency. Moreover, some of fungal and microalgal strains are well known for their ability to treat wastewater, generating biomass which represents a renewable and sustainable feedstock for bioenergy production.ResultsWe screened 33 fungal strains, isolated from compost, straws and soil for their lipid content and flocculation efficiencies against representatives of microalgae commercially used for biodiesel production, namely the heterotrophic freshwater microalgae Chlorella protothecoides and the marine microalgae Tetraselmis suecica. Lipid levels and composition were analyzed in fungal-algal pellets grown on media containing alternative carbon, nitrogen and phosphorus sources from wheat straw and swine wastewater, respectively. The biomass of fungal-algal pellets grown on swine wastewater was used as feedstock for the production of value-added chemicals, biogas, bio-solids and liquid petrochemicals through pyrolysis. Co-cultivation of microalgae and filamentous fungus increased total biomass production, lipid yield and wastewater bioremediation efficiency.ConclusionFungal-assisted microalgal flocculation shows significant potential for solving the major challenges facing the commercialization of microalgal biotechnology, namely (i) the efficient and cost-effective harvesting of freshwater and seawater algal strains; (ii) enhancement of total oil production and optimization of its composition; (iii) nutrient supply through recovering of the primary nutrients, nitrogen and phosphates and microelements from wastewater. The biomass generated was thermochemically converted into biogas, bio-solids and a range of liquid petrochemicals including straight-chain C12 to C21 alkanes which can be directly used as a glycerine-free component of biodiesel. Pyrolysis represents an efficient alternative strategy for biofuel production from species with tough cell walls such as fungi and fungal-algal pellets.

Dual application of duckweed and azolla plants for wastewater treatment and renewable fuels and petrochemicals production

BackgroundShortages in fresh water supplies today affects more than 1 billion people worldwide. Phytoremediation strategies, based on the abilities of aquatic plants to recycle nutrients offer an attractive solution for the bioremediation of water pollution and represents one of the most globally researched issues. The subsequent application of the biomass from the remediation for the production of fuels and petrochemicals offers an ecologically friendly and cost-effective solution for water pollution problems and production of value-added products.ResultsIn this paper, the feasibility of the dual application of duckweed and azolla aquatic plants for wastewater treatment and production of renewable fuels and petrochemicals is explored. The differences in absorption rates of the key wastewater nutrients, ammonium and phosphorus by these aquatic macrophytes were used as the basis for optimization of the composition of wastewater effluents. Analysis of pyrolysis products showed that azolla and algae produce a similar range of bio-oils that contain a large spectrum of petrochemicals including straight-chain C10-C21 alkanes, which can be directly used as diesel fuel supplement, or a glycerin-free component of biodiesel. Pyrolysis of duckweed produces a different range of bio-oil components that can potentially be used for the production of “green” gasoline and diesel fuel using existing techniques, such as catalytic hydrodeoxygenation.ConclusionsDifferences in absorption rates of the key wastewater nutrients, ammonium and phosphorus by different aquatic macrophytes can be used for optimization of composition of wastewater effluents. The generated data suggest that the composition of the petrochemicals can be modified in a targeted fashion, not only by using different species, but also by changing the source plants’ metabolic profile, by exposing them to different abiotic or biotic stresses. This study presents an attractive, ecologically friendly and cost-effective solution for efficient bio-filtration of swine wastewater and petrochemicals production from generated biomass.

Biosynthesis of Proanthocyanidins in White Clover Flowers: Cross Talk within the Flavonoid Pathway

Proanthocyanidins and anthocyanins are produced by closely related branches of the flavonoid pathway and utilize the same metabolic intermediates. Previous studies have shown a flexible mechanism of flux diversion at the branch-point between the anthocyanin and proanthocyanidin pathways, but the molecular basis for this mechanism is poorly understood. Floral tissues in white clover plants (Trifolium repens) produce both proanthocyanidins and anthocyanins. This makes white clover amenable to studies of proanthocyanidin and anthocyanin biosynthesis and possible interactions within the flavonoid pathway. Results of this study show that the anthocyanin and proanthocyanidin pathways are spatially colocalized within epidermal cells of petals and temporally overlap in partially open flowers. A correlation between spatiotemporal patterns of anthocyanin and proanthocyanidin biosynthesis with expression profiles of putative flavonoid-related genes indicates that these pathways may recruit different isoforms of flavonoid biosynthetic enzymes. Furthermore, in transgenic white clover plants with down-regulated expression of the anthocyanidin reductase gene, levels of flavan 3-ols, anthocyanins, and flavonol glycosides and the expression levels of a range of genes encoding putative flavonoid biosynthetic enzymes and transcription factors were altered. This is consistent with the hypothesis that flux through the flavonoid pathway may be at least partially regulated by the availability of intermediates.

Enhanced Biological Straw Saccharification Through Coculturing of Lignocellulose-Degrading Microorganisms

Lignocellulosic waste (LCW) is an abundant, low-cost, and inedible substrate for the induction of lignocellulolytic enzymes for cellulosic bioethanol production using an efficient, environmentally friendly, and economical biological approach. In this study, 30 different lignocellulose-degrading bacterial and 18 fungal isolates were quantitatively screened individually for the saccharification of four different ball-milled straw substrates: wheat, rice, sugarcane, and pea straw. Rice and sugarcane straws which had similar Fourier transform-infrared spectroscopy profiles were more degradable, and resulted in more hydrolytic enzyme production than wheat and pea straws. Crude enzyme produced on native straws performed better than those on artificial substrates (such as cellulose and xylan). Four fungal and five bacterial isolates were selected (based on their high strawase activities) for constructing dual and triple microbial combinations to investigate microbial synergistic effects on saccharification. Combinations such as FUNG16-FUNG17 (Neosartorya fischeri–Myceliophthora thermophila) and RMIT10-RMIT11 (Aeromonas hydrophila–Pseudomonas poae) enhanced saccharification (3- and 6.6-folds, respectively) compared with their monocultures indicating the beneficial effects of synergism between those isolates. Dual isolate combinations were more efficient at straw saccharification than triple combinations in both bacterial and fungal assays. Overall, co-culturing can result in significant increases in saccharification which may offer significant commercial potential for the use of microbial consortia.

Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T0 and T1 generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T4 generation in an agronomically elite ‘Grasslands Sustain’ genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T4 generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.

A high-resolution method for the localization of proanthocyanidins in plant tissues

BackgroundHistochemical staining of plant tissues with 4-dimethylaminocinnamaldehyde (DMACA) or vanillin-HCl is widely used to characterize spatial patterns of proanthocyanidin accumulation in plant tissues. These methods are limited in their ability to allow high-resolution imaging of proanthocyanidin deposits.ResultsTissue embedding techniques were used in combination with DMACA staining to analyze the accumulation of proanthocyanidins in Lotus corniculatus (L.) and Trifolium repens (L.) tissues. Embedding of plant tissues in LR White or paraffin matrices, with or without DMACA staining, preserved the physical integrity of the plant tissues, allowing high-resolution imaging that facilitated cell-specific localization of proanthocyanidins. A brown coloration was seen in proanthocyanidin-producing cells when plant tissues were embedded without DMACA staining and this was likely to have been due to non-enzymatic oxidation of proanthocyanidins and the formation of colored semiquinones and quinones.ConclusionsThis paper presents a simple, high-resolution method for analysis of proanthocyanidin accumulation in organs, tissues and cells of two plant species with different patterns of proanthocyanidin accumulation, namely Lotus corniculatus (birdsfoot trefoil) and Trifolium repens (white clover). This technique was used to characterize cell type-specific patterns of proanthocyanidin accumulation in white clover flowers at different stages of development.

Reduced lignin content and altered lignin composition in the warm season forage grass Paspalum dilatatum by down-regulation of a Cinnamoyl CoA Reductase Gene

C4 grasses are favoured as forage crops in warm, humid climates. The use of C4 grasses in pastures is expected to increase because the tropical belt is widening due to global climate change. While the forage quality of Paspalum dilatatum (dallisgrass) is higher than that of other C4 forage grass species, digestibility of warm-season grasses is, in general, poor compared with most temperate grasses. The presence of thick-walled parenchyma bundle-sheath cells around the vascular bundles found in the C4 forage grasses are associated with the deposition of lignin polymers in cell walls. High lignin content correlates negatively with digestibility, which is further reduced by a high ratio of syringyl (S) to guaiacyl (G) lignin subunits. Cinnamoyl-CoA reductase (CCR) catalyses the conversion of cinnamoyl CoA to cinnemaldehyde in the monolignol biosynthetic pathway and is considered to be the first step in the lignin-specific branch of the phenylpropanoid pathway. We have isolated three putative CCR1 cDNAs from P. dilatatum and demonstrated that their spatio-temporal expression pattern correlates with the developmental profile of lignin deposition. Further, transgenic P. dilatatum plants were produced in which a sense-suppression gene cassette, delivered free of vector backbone and integrated separately to the selectable marker, reduced CCR1 transcript levels. This resulted in the reduction of lignin, largely attributable to a decrease in G lignin.

An improved method for quantitative analysis of total fructans in plant tissues.

Current methods for measuring fructan levels in plant tissues are time-consuming and costly. They often involve multiple or sequential extractions, enzymatic or acid hydrolysis of fructan polymers, and multiple HPLC runs to quantify fructan-derived hexoses. Here we describe a new method that requires a single extraction step, followed by selective precipitation of fructans by acetone, acid hydrolysis of the precipitate, and a short (10 min) HPLC run to complete the procedure. We used perennial ryegrass samples to show that the new method has similar sensitivity, but better reproducibility, than a more complex method that is widely used. We have used the new method to study developmentally related changes in fructan levels in glasshouse-grown perennial ryegrass plants.