The aim of the work presented in this thesis was to investigate different mechanical and chemical pre-treatments which can dramatically change the properties of native cellulose and add alternative routes to structure formation. Ball milled cellulose, which had a reduced crystallinity, degree of polymerisation and degradation temperature, was rehydrated in excess water resulting in recrystallisation. Fully amorphous samples recrystallised to the more thermodynamically stable type II polymorphic crystal structure. Flash differential scanning calorimetry (DSC), which allows thermal transitions to be scanned at much higher rates than conventional DSC, was able to register a glass transition temperature for amorphous cellulose. The next stage of the study focussed on the production of freeze dried galactomannan foams. Cellulose fibres provided reinforcement to the foams. The level of reinforcement was related to fibre content, size, crystallinity and surface roughness. Microfibrillated cellulose (MFC) provided the greatest reinforcement due to its much higher surface area and fibrillated structure. Extrusion was found to be a useful alternative to homogenisation for the production of MFC and to create foams using alternative processing to the freeze drying routes. A novel molten salt hydrate, LiCl/urea/water, was found to swell native cellulose and reduce its crystallinity. A weak gel-like structure was formed at ambient temperature. Micro DSC results showed that this structure was melted out at 60oC but the process was reversible indicating hydrophilic to hydrophobic conformational changes on the surface of the cellulose fibres, although these were likely to be dependent on the celluloses having a low degree of polymerisation. In these solvent conditions starch granules were eroded from the outside rather than being swollen as has been found for some ionic liquids and underwent total dissolution in LiCl/urea/water. Fenugreek and xyloglucan, which are both highly branched, were found to increase in viscosity in LiCl/urea/water relative to water, possibly due to the breakage of all intramolecular associations whereas the viscosity of konjac which is predominantly unbranched did not change. Locust bean gum (LBG) had a lower viscosity in LiCl/urea/water compared to water due to the disruption of aggregates. Confocal microscopy showed that fenugreek and LBG are able to bind to cellulose in water, however, the conformational change of fenugreek in these solvent conditions inhibited it from binding to cellulose in LiCl/urea/water whereas conformational change allowed xyloglucan to bind to cellulose in LiCl/urea/water whilst it was unable to bind in water. Konjac did not bind to cellulose in either solvent system. The pre-treatments shown in this work will enable the creation of novel cellulose composites.

Sauropus androgynus, commonly known as ‘sweet shoot’ is an underutilized crop known for its high nutritive values and medicinal properties. To date, scientific studies assessing the potentially important benefits of sweet shoot for use as medicinal plants are still limited, with only six studies reporting on in vitro propagation and nine articles describing the production of secondary metabolites. The present study revealed that shadehouse-grown plants contained a low yield of bioactive phytochemicals, such as phenolic (61.20 µg/10g FW), flavonoid (193.62 µg/10g FW), naringenin (128.01 µg/10g FW), quercetin (1.56 µg/10g FW) and kaempferol (274.85 µg/10g FW). Moreover, it also had very low antioxidant activity in DPPH (54.03%) and FRAP (397.56 µg/10g FW) assay. The elicitation of cultured tissues is therefore necessary to improve the production of phytochemical compounds and to increase the antioxidant capacity in sweet shoot. In this study, four different types of cultured tissues (in vitro shoot cultures, light-induced callus, dark-induced callus and somatic embryos) were selected to achieve this goal, followed by the extraction of phytochemicals from these cultured tissues treated with elicitors and precursor for better production of phytochemicals. For in vitro shoot induction, nodal explants cultured on semi-solid MS medium supplemented with 2.0 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l indole-3-acetic acid (IAA) produced the highest number of shoots (7 shoots per explant) with longer shoot length (5.74 cm). For light-induced callus induction, leaf explants grown in illuminated conditions with semi-solid MS medium enriched with 2.0 mg/l α-naphthalene acetic acid (NAA) and 1.0 mg/l kinetin showed good proliferation from the leaf explants (71.67%) with the highest callus fresh weight (4.53 g) and highest callus expansion rate (18.50 cm2). Likewise, leaf explants induced in dark condition on semi-solid MS medium containing similar hormone composition, also displayed the highest callus fresh weight of 4.54 g and callus expansion rate of 13.85 cm2. Three-month-old dark-induced callus were transferred onto liquid MS medium fortified with a different concentration of NAA and kinetin to further induce somatic embryos. After three weeks of callus inoculation, as high as 83.33% of embryogenic cell cultures achieved its maximum density of 5.2 ml in liquid MS medium supplemented with 2.0 mg/l NAA and 1.0 mg/l kinetin. In histodifferentiation medium (liquid MS medium containing 1.0 mg/l NAA and 0.5 mg/l kinetin), a mean number of 15.60, 14.80 and 13.20 embryos per g callus of globular, heart-shaped and torpedo-shaped embryos developed respectively after 9 weeks of embryo induction. The torpedo-shaped embryos were then inoculated into hormone-free MS medium and 90% of these embryos successfully differentiated into cotyledonary embryos after 3 weeks of maturation. These results showed a complete ontogeny of sweet shoot somatic embryo from the globular stage to heart-shaped, torpedo-shaped and cotyledonary stage. After two months of shoot initiation on MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l IAA, a relatively high percentage (>75%) of shoot proliferation occurred from nodal derived shoot (6.74 shoots per explant), light-induced callus (6.23 shoots per callus) and somatic embryos (6.45 shoots per embryo) of sweet shoot. These well proliferated shoots were then subjected to root initiation in half strength MS medium containing 1.0 mg/l IAA and high percentage of root formation was successfully achieved in 90% of the plantlets after 10 days of culture. For hardening off, the rooted plantlets were transferred to culture jars containing purified water and maintained at ambient conditions for one month. High survival rate (>76.67%) was achieved in perlite:compost mixture (1:1) after one month of acclimatization in shadehouse. To enhance the production of phytochemicals and antioxidant capacity in tissue cultures of sweet shoot, elicitor and precursor treatments served as alternative methods in influencing the biosynthetic pathway for the accumulation of phytochemicals. In this study, shadehouse-grown plants and cultured tissues of sweet shoot were treated individually with different concentrations of methyl jasmonate (MJ), salicylic acid (SA) and phenylalanine (Phe) for a treatment period of 3 weeks. Light-induced callus culture produced the highest amount of phenolic and flavonoid compounds amongst the tested plant samples with those treated with Phe producing the highest antioxidants, followed by MJ and SA. After 3 weeks of Phe treatment at 20 mg/l, the highest levels of total phenolic (246.62 µg/10g FW), total flavonoid (636.26 µg/10g FW), naringenin (12081.05 µg/10g FW), quercetin (134.36 µg/10g FW), kaempferol (11325.13 µg/10g FW) and antioxidant activities (97.35% for DPPH and 5941.66 µg/10g FW for FRAP assay) were detected in light-induced callus cultures of sweet shoot. Since phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) were the key enzymes for the biosynthesis of phenolics and flavonoids, both enzymatic activities were also measured in light-induced callus cultures treated with Phe. The highest PAL (101.18 mmol CA/g FW) and CHS (14.49 nkat/mg protein) enzymatic activities were also attained in light-induced callus cultures fed with 20 mg/l of Phe at week 3. Light-induced callus cultures treated with Phe produced the highest amounts of phytochemicals, antioxidant capacity and enzymatic activities, and these results were chosen to undergo Pearson’s correlation coefficient analysis, which verified the positive co-relationship seen between all of the above-mentioned parameters. These findings showed that the addition of Phe enhanced the enzymatic activities in the phenylpropanoid pathway and increased the concentrations of phenolic and flavonoid compounds (naringenin, quercetin, kaempferol) which in turn contributed to the increase in antioxidant activities in light-induced callus cultures of sweet shoot. Data from this study showed that sweet shoot has the potential to be developed as a plant-based antioxidant for the pharmaceutical industry. Additionally, this study is the first to report on the complete ontogeny of sweet shoot and the positive effects of elicitation in tissue cultures of sweet shoot.

The aim of this work was a reinvestigation of the role of Cl('-) ions in the reactions on the oxidizing side of photosystem II in chloroplasts. It was found that thylakoid membranes retained photosynthetically active Cl('-) during their isolation in Cl('-)-free media. This Cl('-) was readily released only when the thylakoids were incubated in uncoupler-containing media at alkaline pH in the dark. Once the residual Cl('-) was released by such treatment, photosynthetic electron transport became dependent on added Cl('-) under all conditions. / The effect of Cl('-) deficiency on the stability of the higher oxidation states of the Mn-containing water photooxidase was investigated. While at least two oxidizing equivalents were accumulated on this enzyme in the absence of Cl('-), they were not as stable in the dark as when Cl('-) was present. The postulate that Cl('-) serves only as a counter ion to positively charged oxidation states of the water splitting enzyme could not be supported. / In addition to facilitating the release of Cl('-) from thylakoids, uncouplers were found to also accelerate the release of bound Mn from PS II in dark-adapted chloroplasts. This suggested that a metastable proton gradient could persist in thylakoids form some time in the dark. It is postulated that both the photosystem II Cl('-) site and the water splitting enzyme reside in a special proton sequestering domain located within the thylakoid membrane which is isolated from the bulk solutions by barriers that are nearly impermeable to protons. The experimental evidence suggests that this domain may be identical to that postulated by Dilley's research group (Baker, G. M., Bhatnager, D. and Dilley, R. A., 1980, Biochemistry 20: 2307-2315). / Source: Dissertation Abstracts International, Volume: 42-09, Section: B, page: 3520. / Thesis (Ph.D.)--The Florida State University, 1981.

The curds of cauliflower (Brassica oleracea var.botrytis L.) which are representative of the European biennials, European annuals and Australian types were used to extract aspartate aminotransferase (EC 2.6.1.1) and acid phosphatase (EC 3.1.3.2). These enzymes were separated into their isoenzymes using polyacrylamide gel electrophoresis and were shown to have different numbers of isoenzymes depending upon which of the three main groups of cauliflower cultivars were used. The enzymes examined showed evolutionary divergence of the cauliflower types. Growth and development, storage and frost damage in cauliflowers were examined in relation to peroxidase activity. Physiological age in different parts of the curds and between different size curds was shown to have very little effect on peroxidase activity. Total peroxidase activity in the stem was about 2 times the value in the curds. A gradual increase of peroxidase was found to occur with duration of storage period at 7°C. Peroxidase activity remained low when cauliflowers were kept at low temperatures, namely both -1 + 1°C and -5 + 1°C, but increased rapidly on warming up at 7°C for 2 days. Isolation and partial purification of the peroxidase were carried out using conventional methods, including ammonium sulphate precipitation, Sephadex G-100 molecular sieve and DEAE Cellulose ion exchange chromatography. Further purification was achieved using Phenyl Sepharose hydrophobic interaction chromatography. Peroxidase activity was localized in the 50-95% ammonium sulphate precipitation. Molecular sieve chromatography showed a broad peak of peroxidase activity and molecular weight was estimated to be about 42,000. Polyacrylamide gel electrophoresis and isoelectric focusing of this fraction showed the existence of two peroxidase isoenzymes with P 6.7 and 7.3 using guaiacol as substrate; one of the isoenzymes also possessed a simultaneous IAA oxidase activity. Optimal pH and temperature with guaiacol as a substrate was found to be pH 5.6 and 50°C. Kinetic studies indicated an apparent Km of 8.3 mM for guaiacol at an optimal hydrogen peroxide concentration and a Km of 6.25 mM for hydrogen peroxide at an optimal guaiacol concentration. By including hydrophobic interaction chromatography in the purification scheme, a homogeneous cauliflower peroxidase isoenzyme was obtained. It showed as a single band in polyacrylamide gel electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Molecular weight estimation of this peroxidase isoenzyme was 41,000+1,000. The elution profiles of white and green cauliflowers and white sprouting broccoli peroxidases by hydrophobic interaction chromatography were found to be different. Green cauliflowers with high frost resistance were found to have high percentage of peroxidase activity bound very tightly to the hydrophobic resin. Similar peroxidase activity patterns using guaiacol, o-dianisidine and pyrogallol were obtained. Antiserum specific for the homogeneous peroxidase from cauliflower (cv. Milreef) was used to examine homology among cauliflower peroxidases and among peroxidases from green and white cauliflowers, white sprouting broccoli and horseradish. The antiserum cross reacted with all cauliflower peroxidases as well as peroxidases from green cauliflower, white sprouting broccoli and horseradish, indicating the antigenic sites were similar.

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Lateral roots are a key component of the plant root system architecture that help anchorage in soil and acquisition of water and nutrients. In the dicot Arabidopsis, lateral roots initiate post-embryonically from a specialised set of cells at the xylem pole of the pericycle cell layer termed ‘founder cells (FC)’, overlaid by three distinct tissue layers – endodermis, cortex, epidermis. The FCs undergo a coordinated series of asymmetric cell divisions (ACD) to form a primordium that grows and emerges through these overlying layers as a mature lateral root. Different auxin signaling modules, as well as tight regulation of the cell geometry are important during early organogenesis. In this study, we were interested to identify molecular components that influence cell wall remodeling properties and cell geometry in the FC and regulates asymmetric cell division during early lateral root initiation. Transcriptomic analysis of FCs (De Smet et al., 2008) identified a candidate gene EXPANSINA1 (EXPA1) of expansin superfamily, known for their unique ability to alter linkage between the cell wall polymers and cause wall loosening. In vivo expression studies showed that EXPA1 is expressed in FCs prior asymmetric cell division. The mutant expa1-1 exhibits perturbed ACD with a delay in kinetics of primordia from Stage I to II, loss in radial expansion of FCs in response to auxin which is important for organised formative divisions. To understand if these defects are due to altered properties of the cell wall, and role of auxin in this process, we developed an optimised technique to study the chemical properties of the FC cell wall junctions based on confocal Raman spectroscopy. This showed altered interactions in expa1-1 between the major cell wall polymers xyloglucans and pectins locally in the pericycle cell wall upon auxin treatment, that could influence cell geometry during early lateral root development. Additionally, sugar monomer analysis of digested whole roots showed interesting alterations in representative global wall sugar levels in the root which although diluted due to lack of tissue specificity warrants further study. In conclusion, the combination of molecular and biochemical analyses reveals that auxin dependent regulation of EXPA1 plays an important role in lateral root FC and is required for organised asymmetric cell division.

The many advantages of light-emitting diode (LED) grow lamps in terms of programmability and energy efficiency make them an attractive replacement for old lighting systems. However, when replacing conventional broad-waveband ‘white’ lighting with narrow-waveband LED lighting it is important to ensure that the system is able to produce plants of an equal or higher quality and yield. In order to choose suitable light ‘recipes’, i.e. combinations of wavelengths for optimal plant growth, we must first understand how light quality affects plant growth and development. The following work explores the role of each part of the light spectrum, both in natural sunlight in canopy environments in the field and glasshouse, and in an artificial light environment in controlled environment growth rooms. It was determined that, blue, green, red and far-red wavebands are all instrumental in the provision of positional signals to the leaf which enable optimisation of plant physiology to light conditions, especially in the canopy environment. A new hypothesis for the role of the blue:green light ratio in whole-canopy water use efficiency is presented. Wavelength-specific effects were observed in hypocotyl elongation, leaf expansion, photosynthetic stoichiometry and absorption of different wavelengths, root architecture, photosynthetic capacity, whole-plant morphology (in terms of biomass partitioning between leaves, stems and roots) and fresh weight yield of stems and leaves. In the glasshouse, it was determined that monochromatic red LED lighting is a suitable alternative for conventional high-pressure sodium light, as it boosted leaf photosynthetic capacity as well as leaf area, fresh and dry weight yield, and was the most efficient light source in terms of ‘crop per watt’. Finally, further applications of the work such as those for commercial horticulture and farming in closed plant production systems are presented, and it is concluded that LED lighting is likely to have a crucial role in the future of global food security.

Tomography (CT) offered a distinctive method for visualising undisturbed RSA which was used to develop an understanding of the response of wheat (Triticum aestivum L.) to compaction. The first aim was to assess the impact of soil properties and X-ray scanning parameters have on image quality, and identify which settings are optimum for balancing against acquisition time. The second aim was to investigate how soil compaction affects the growth and RSA of wheat, while also considering the impact of soil texture. The third aim was to test if and how soil compaction alters a roots mechanical properties, and what anatomical changes are responsible for these changes. Using complementary techniques including root tensile strength testing and root sectioning, insights have been gained on the anatomical adaptions of roots and how these changes affect their material properties and behaviour in compacted soil. This will allow more efficient phenotyping of wheat varieties for soil exploration and resource uptake.

Lateral roots (LR) contribute considerably to the architecture of the root system. The hormone auxin tightly controls the regulation of LR formation in response to environmental signals. For example, roots have the ability to distinguish between wet and dry micro environments in the soil and adapt the positioning of lateral roots accordingly. This concept is referred to as LR hydropatterning and is a novel adaptive mechanism for controlling root branching. When growing vertically down an agar plate, Arabidopsis thaliana roots are also exposed to an asymmetric distribution of water that causes a meniscus to form around the primary root (PR) circumference. LRs develop preferentially on the side of the PR in contact with water, rather than the side exposed to air. My project aims to elucidate the underlying molecular mechanisms controlling this novel adaptive response. It has been revealed that the transcription factor AUXIN RESPONSE FACTOR 7 (ARF7) is essential for LR hydropatterning. In contrast to wild type, arf7 loss of function mutants do not exhibit greater LR emergence on the side of the PR in contact with moisture. Ectopic expression of ARF7 (in 35S:ARF7 arf7-1) can rescue arf7 LR hydropatterning, implying that ARF7 regulates LR hydropatterning via a post-transcriptional mechanism. One promising post-transcriptional mechanism that may control LR hydropatterning involves protein SUMOylation (a Small Ubiquitin-like Modifier), since the SUMO mutant ots1 ots2 phenocopies the arf7 LR hydropatterning defect. OTS1 and OTS2 encode nuclear localised proteases that remove SUMO from target proteins. ARF7 is a target for SUMO modification, containing several SUMOylation sites including one within its DNA binding domain. Expressing wild type ARF7 and a non-SUMOylated version in arf7 revealed that ARF7 controls LR hydropatterning via SUMOylation. This knowledge will help reveal how plants explore the soil and position LR to maximise water (and nutrient) foraging.

Plants need to defend themselves against their natural enemies without compromising their interactions with beneficial organisms. Chemical mechanisms underpin many of these interactions and changes in plant metabolism are critical to both robust defences against antagonists and effective signals to mutualists. Further, such plant responses can be systemic, so mediating interactions between spatially separated organisms above and below ground. This thesis aimed to characterise the changes in the ragwort (Senecio jacobaea L.) metabolome caused by two different belowground organisms, an antagonistic herbivorous nematode (Pratylenchus penetrans (Cobb, 1917) Filipjev & Schuurmans Stekhoven, 1941) and a mutualist arbuscular mycorrhizal fungus (AMF) (Glomus intraradices Smith & Schenck). Initially, vegetative and reproductive stage ragwort plants were sampled in the field and the chemical composition of leaf and flower tissues was assessed using a metabolomic approach. Techniques for the identification of key ragwort secondary metabolites were trialled and results demonstrated that plants of different ages differed in their allocation of within plant defences such as flavanoids, pyrrolizidine alkaloids (PAs) and chlorogenic acids. Subsequent experiments with nematodes and AMF focussed on the analysis of leaf tissues from vegetative stage plants. Feeding by the nematode species P. penetrans resulted in increased concentrations of metabolites associated with plant defence, including the main class of ragwort defence compounds PAs. In contrast, colonisation of root material by AMF caused increases in the concentrations of metabolites associated with the maintenance of the beneficial interaction between plant and fungi, such as a number of apocarotenoids known as blumenols. The findings of both experimental studies detected unexpected and previously unreported changes in plant metabolism, highlighting the importance of an untargeted approach when examining the chemical ecology of plant interactions.