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Lignocellulosic biomass, composed largely of plant cell walls, of economically important cereal crops is remarkably recalcitrant to digestion, both in second generation biofuel production and ruminant nutrition applications. Ferulic acid (FA) esterified to arabinoxylan (AX) forms oxidatively linked dimers and oligomers which cross-link polysaccharide chains. FA is also a nucleation site for lignin formation. These cross-links are major inhibitors of enzymatic digestion, and therefore FA is a key target for improving the digestibility of grass cell walls. Also, para coumaric acid (pCA) esterified to AX may be involved in the polymerisation of lignin. Despite the importance of cell wall bound hydroxycinnamic acids, many of the genes and enzymes responsible for the esterification of pCA and FA to AX remain to be elucidated. The BAHD and glycosyltransferase (GT)61 gene families have previously been identified as likely to be involved in the process (Mitchell et al., 2007). Here, the role of candidate genes within the BAHD and GT61 families in pCA and FA esterification to AX is investigated in the model organism Brachypodium distachyon (Brachypodium). Jasmonic acid induced large increases in cell wall-esterified pCA, and moderate increases in FA and FA dimer in Brachypodium callus, accompanied by up-regulation of genes within the BAHD and GT61 families. Furthermore, transformation of Brachypodium with RNAi constructs designed to knock-down expression of paralogues BdGT61.9p1 and BdGT61.9p2 resulted in decreased cell wall-esterified FA. Overexpression of BdGT61.9p1 in Brachypodium resulted in a small increase in the 8-8-coupled FA dimer. These findings complemented the existing body of evidence for the involvement of genes within the BAHD and GT61 families in hydroxycinnamic acid esterification to AX.

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No description available.

The domestication of plants and animals has been a powerful force in the development of human societies over the past millennia. Domestication of plants is underscored by the selection of agriculturally favourable traits, such as flowering time and disease resistance, which are often inherited in a quantitative manner. Advances in techniques relating to the study of quantitative traits over the past decades enable the dissection of the genetic architecture and molecular basis of these traits. In this thesis, I discuss the natural diversity governing flowering time and intermediate nonhost resistance in the non-domesticated grass Brachypodium distachyon. Three major loci were found to govern flowering time, two of which colocalise with the B. distachyon homologs of major flowering pathway genes identified in crop species. However, the identification of additional loci suggests that greater complexity underlies flowering time in this non-domesticated system. In contrast, a natural stack of three resistance genes protects B. distachyon against colonisation by Puccinia striiformis and highlights a relatively simple genetic architecture for intermediate nonhost resistance. One broad spectrum major effect locus was narrowed down to genes that are commonly associated with isolate-specific host resistance While it has been proposed that nonhost and host resistance are inherently different, the genetic architecture and molecular basis of resistance in this intermediate nonhost system is reminiscent of a host system, which suggests that the genetic architectures of host and nonhost systems are structurally coupled and share conserved components. Studying the genetic basis of these quantitative traits in B. distachyon elucidates the way humans have utilised the natural variation present in grasses to create modern temperate cereals. Additionally, exploring the interaction between B. distachyon and P. striiformis has provided an ideal system to investigate the transfer of resistance genes from wild relatives to agronomically important crops.

Bluebell chemistry was investigated in order to identify factors contributing to the success and survival of the plant in a bluebell-bracken dominated ecosystem. The plant was divided into roots, bulbs, leaves, scapes and flowers and three classes of compounds (carbohydrates, phenolics and saponins) were studied and their seasonal changes were followed. Total non-structural carbohydrates (TNC) were quantified after acid hydrolysis of the dried plant material and analysis of the reaction product by high performance liquid chromatography with diode array detection (HPLC-DAD). The bulbs contained the highest carbohydrate content compared to the other parts with TNC levels ranging from 36 – 82% of dry weight. Highest values in bulbs were reached around anthesis and remained high until the end of above ground growth. The lowest values were recorded during the underground growth phase before shoot emergence in March. The above ground parts showed low TNC contents at the start of their growth and higher values were reached during the active above ground growth. Mono- and disaccharides were studied using gas chromatography-mass spectrometry (GC-MS) after conversion into volatile oxime-trimethyl silyl derivatives. Fructose, glucose and sucrose were detected in this pool with fructose being the major sugar in the underground organs versus glucose in the above-ground parts. The bulbs showed significant increase in monosaccharides, especially fructose, during the early growth. Fructans were also investigated and their amounts were determined in the above and below ground organs following an enzymatic assay. The results showed a trend similar to the TNC especially in the bulbs. The fructan pool was further studied with matrix-assisted laser desorption ionisation – time of flight (MALDI-ToF) spectroscopy to determine the chain length of the polymer. This revealed that short chains < 10 units are more commonly found with DP3 and DP4 being the most abundant oligomers. Higher chain lengths (up to DP20) was also found, but in much lower quantities. The main trisaccharide was isolated using flash chromatography and preparative TLC in reverse phase mode. Structural elucidation was achieved using 1D and 2D NMR spectroscopy of the free and peracetylated forms and the trisaccharide was identified as 1-kestose. The phenolic profile of bluebell flowers, scapes and leaves was thoroughly studied using HPLC-UV and ESI-MS/MS analysis. The results showed that apigenin was the main flavone aglycone in the plant in addition to minor quantities of luteolin, eriodycteol and other unidentified aglycones. Phenolic acids including p-coumaric and ferulic acids were also detected. Both O- and C-glycosides were detected. The glycosidic moieties identified as hexoses, pentoses, rhamnose and glucuronic acid. The methanolic extract of bluebell flowers was investigated and four new metabolites were isolated including two phenylalkyl glycosides (benzyl-O-β-D-glucopyranosyluronic acid-(1→2)-glucopyranoside DR1 and 2-phenylethyl-O-β-D-glucopyranosyluronic acid-(1→2)-glucopyranoside DR2) and two apigenin glycosides (7-[O-β-D-glucopuranosyl-(1→3)-O-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→2)-O-β-D-glucglucopyranoside uronic acid-(1→)] apigenin (DR3) and 7-[ O-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→2)-O-β-D-glucglucopyranoside uronic acid-(1→)] apigenin (DR4)). The structural elucidation of these compounds was based on NMR, accurate mass and MS/MS analysis. Apigenin and p-coumaric acid were the only aglycones quantified after acid hydrolysis of the hydrolysed dried plant. The results showed that the two aglycones accumulated to a higher extent in the above ground organs with anthesis being characterised with an increase in content of both compounds to up to 15 and 0.5 mg. g-1 DM, respectively. A previously unreported saponin, (3β-[(O-β-D-glucopyranosyl-(1→3)-O- β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranosyl-(1→2)-O-α-L-arabinopyranosyl-(1→6)-O-β-D-glucopyranosyl)oxy]-17,23-epoxy-28,29-dihydroxy-27-norlanost-8-en-24-one (DR1), was isolated from bluebell flowers using RP flash chromatography. The structure was elucidated with extensive 1D and 2D NMR, acid hydrolysis and GC-MS analysis of the sugars, accurate mass and MS/MS analysis. The isolated saponin was investigated for potential anti-trypanosomal, antimicrobial, antifungal, pesticidal and schistosomicidal activities. DR1 showed high biological activities against Trypanosoma brucei and Schistosoma mansoni. Crude fractions (aqueous and 1-butanol fractions of the methanolic extracts) from bluebell seeds were also investigated for schistosomicidal activities and found to show comparable results to DR1. The metabolomics approach and multivariate analysis approach was used in order to investigate the saponins and follow their seasonal changes. The seasonal samples of different plant parts were analysed with LC-MS in positive and negative modes. The resulting spectra were processed and the data produced were compared to databases for possible identification of metabolites. Multivariate analysis highlighted the difference between bulb metabolites from the above ground organs. The LC-MS spectra obtained and peak data used to study the changes in saponin contents between the different organs throughout the study period. The bulbs were found to contain the lowest amounts of total saponins (only about 20% of the other organs) with the highest ratios being in March-April at the start of the growth then decrease afterwards. Amongst the above-ground organs, the flowers showed relatively higher contents. The LC-MS spectra from the metabolomics study were utilised in tentatively identifying a number of saponins. Two other metabolites were identified as steroidal aglycones from their HR-ESI-MS spectra. These metabolites were of 470 and 456 mass units and were characteristic of the bulbs.

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No description available.

Investigations were made into the behaviour of wheat tissues in vitro, with regard to the initiation of callus and its subsequent growth and morphogenesis, and with particular emphasis upon defining the factors leading to plant regeneration. Despite quantitative genotypic differences, consistent callus initiation was possible from all parts of the embryo (mature and immature), the young seedling and mature plant, with the excepation of the scutellum, the expanded leaf blade and certain segments of internode (endosperm cultures were not tested). Auxin was essential for callus initiation and growth; the concentration of 2,4-D required for optimum yield of callus depended on the source of explant. Primary cultures had slightly higher auxin requirements than older callus. Typically the primary callus grew rapidly, but increase in fresh weight declined during subsequent passages. Use of several different media had little effect on growth. The time for the culture to double its biomass ("doubling-time") was affected by passage length, hormone concentration and age of the culture. Addition of cytokinins was found to elicit little response other than a slight increase in rhizogenesis. Rhizogenesis was readily stimulated by decreasing the auxin level, but shoot formation was sporadic. A low proporation of shoot-bearing cultures was observed in many separate experiments, under cultures was observed in many separate experiments, under different conditions of hormone treatment and using callus derived from explants of many sources. Shoot formation was more common during the second passage, and occurred either as a single clump from within the callus, or (more rarely) as superficial leafy outgrowths. The former type of shoot formation is suggested to be due to expression of auxin-inhibited primordial, originating from the explants, continuing development once auxin is depleted. Histological examinations revealed the source of callus initiation from different types of explants, and showed the internal structure of organogenetic cultures. Wheat cultures grew well in liquid medium, but as aggregates greater than 1.0 mm diameter; aggregation was little affected by the level of auxin in the medium. In liquid medium, rhizogenesis and greening were the only types of differentiation observed. A project of limited soope inovolving study of the behaviour of uninucleate pollen in cultured wheat anthers demonstrated the low incidence of further mitosis and the rapid loss of pollen viability.

No description available.