Retracing the molecular basis and evolutionary history of the loss of benzaldehyde emission in the genus Capsella

The transition from pollinator‐mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate‐CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer Capsella orientalis. We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found within and outside of Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent

Elucidating the Biosynthetic Routes and Biological Mechanisms Involved in the Release of Phenylpropanoid/Benzenoid Volatiles from Plant Cells

Sessile organisms, such as plants, have developed intricate means of responding and interacting with their environment in order to grow, reproduce, and survive environmental stresses such as attacks by other organisms and competition for resources with neighboring organisms. Plant volatile organic compounds (VOCs) play vital roles in resolving these evolutionary constraints associated with the sedentary nature of plants by attracting pollinators and seed dispersers necessary for reproduction. VOCs also mediate plant-plant interactions and provide defense against biotic stresses (pathogens, predators, and herbivores) and abiotic stresses (oxidative stress, high temperature stress, drought). Beyond the importance of VOCs to plants, humans have used VOCs for centuries as perfumes, therapeutics, food additives, and contribute to the flavor and aroma of fruits and vegetables. Chapter 1 of this dissertation offers a brief overview of plant VOCs, their functions, and biosynthetic pathways. The factors influencing their emission and the long-standing diffusion model of volatile release from plant cells are also discussed. Chapter 2 challenges the diffusion model and provides evidence for the involvement of active transport in the passage of VOCs across the plasma membrane. Downregulation of an ATP-binding cassette (ABC) transporter, PhABCG1, by RNA interference (RNAi) in Petunia hybrida flowers led to a decrease in volatile emission and accumulating of the internal pools of volatiles to toxic levels in the plasma membrane. In addition, PhABCG1 was shown to directly transport benzenoid volatiles using Nicotiana tabacum BY2 cells overexpressing PhABCG1. Together, these results alter the default assumption that VOCs simply diffuse out of cells. Chapter 3 completes the identification of the biosynthetic genes in the peroxisomal β-oxidative pathway of benzoic acid (BA) biosynthesis by the identification of a petunia cinnamoyl-CoA hydratase-dehydrogenase (PhCHD). Kinetic analysis of recombinant PhCHD shows that it converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA in vitro. Furthermore, downregulation of PhCHD in petunia flowers, using an RNAi approach, led to a decrease in benzoyl-CoA (BA-CoA), BA and other benzenoid derived volatiles further demonstrating the involvement of this gene to the peroxisomal β-oxidative pathway of BA biosynthesis. Lastly, Chapter 4 investigates the possible mechanisms of transport of the final product BA-CoA, of β-oxidative BA metabolism out of peroxisomes. Since the CoA moiety is membrane impermeable and BA-CoA thioesterase activity is enriched in purified peroxisomes, we hypothesized that BA-CoA is converted to BA by a thioesterase prior to transport or diffusion across the membrane and then reconverted to BA by a CoA ligase such as Ph4CL1 (petunia 4-coumarate: CoA ligase 1) or BZL1 (benzoate: CoA ligase) or by an unknown membrane associated ligase. Characterization of recombinant PhTE1 shows that it efficiently converts several hydroxycinnamoyl-CoA thioesters, including BA-CoA, to their free acids. Also, downregulation of PhTE1 led to an increase in the levels of BA-CoA and its derived volatiles, suggesting that BA-CoA is most likely transported out of peroxisomes. Furthermore, the levels of volatile phenylpropenes, anthocyanin and lignin were also altered suggesting cross-talk between the ?-oxidative and the general phenylpropanoid pathways. Together, these results suggest the auxiliary roles thioesterases play in β-oxidative metabolism

Biodegradation of Pollutants in Waste Water from Pharmaceutical, Textile and Local Dye Effluent in Lagos, Nigeria

Background. Discharged effluents from industry have been responsible for the deterioration of the aquatic environment in many parts of the world, especially in developing countries. Increasing industrialization and urbanization have resulted in the discharge of large amounts of waste into the environment, resulting in high pollution loads. Utilization of microbes such as fungi and bacteria have been used for pollution degradation. Objectives. The aim of this research was to utilize microbial agents such as fungi and bacteria to reduce pollutant loads such as heavy metals in effluent samples. Methods. Three types of effluent (pharmaceutical, textile effluent, and dye) were obtained from Surulere in Lagos Metropolitan Area, Nigeria. Heavy metals analysis was carried out using a flame atomic adsorption spectrophotometer according to standard methods. Samples were cultured for microbes and identified. Bacteria samples were inoculated on nutrient agar and incubated at 37°C for 24 hours. Fungi counts were carried out using potato dextrose agar and incubated at 28°C for 3–5 days. The isolated organisms were identified based on their morphological and biochemical characteristics. Then 100 mL of the effluents was dispensed into 250 mL flasks, and the pH of the medium was adjusted to 7.2 by the addition of either sodium hydroxide or hydrogen chloride and autoclaved at 121°C for 15 minutes. The autoclaved flask was inoculated with 1 mL of bacteria and fungi for 21 days and pH was recorded properly every 48 hours. Results. The results of the physicochemical parameters indicated that conductivity, total suspended solids, total dissolved solids, turbidity, chemical oxygen demand and biochemical oxygen demand for all the three industrial effluents were higher than the World Health Organization (WHO) permissible limits. Heavy metal analysis results show that the effluents had high values for cadmium, above the WHO limit of 0.003 mg/L. Concentrations of zinc ranged from 0.136–1.690 mg/L, and nickel ranged between 0.004–0.037mg/L for the three effluents, within the WHO limit. The identified bacteria were Bacillus subtilis, Klebsiella pneumonia, Salmonella typhi and Bacillus cereus and isolated fungi were Aspergillus flavus and Penicillium chrysogenum. All the physicochemical parameters and heavy metal concentrations were reduced after the biodegradation study in the effluents. Conclusions. The responses observed in the various microbes indicated that the use of microbes for the reduction of environmental pollutants has an advantage over the use of other methods because it is environmentally friendly, low cost, and no new chemicals are introduced into the environment. This method should be encouraged for pollution reduction to bring about ecosystem sustainability advocated for Ghana. Competing Interests. The authors declare no competing financial interests

Emission of volatile organic compounds from petunia flowers is facilitated by an ABC transporter.

Plants synthesize a diversity of volatile molecules that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. Despite progress in deciphering plant volatile biosynthesis, their release from the cell has been poorly understood. The default assumption has been that volatiles passively diffuse out of cells. By characterization of a Petunia hybrida adenosine triphosphate-binding cassette (ABC) transporter, PhABCG1, we demonstrate that passage of volatiles across the plasma membrane relies on active transport. PhABCG1 down-regulation by RNA interference results in decreased emission of volatiles, which accumulate to toxic levels in the plasma membrane. This study provides direct proof of a biologically mediated mechanism of volatile emission