Species-rich grasslands have seen a drastic decline due to agricultural intensification and abandonment and this has resulted in a fragmented distribution of grassland sites. Such grasslands are highly diverse and are being managed for conservation. Long-term studies provide valuable evidence for the effective management of grasslands and this research investigated how the vegetation of species-rich grasslands in the Forest of Bowland, NW England has changed over 25 years, a period which has seen the introduction of statutory protection measures and agrienvironment schemes. The study examined change in 35 grassland sites under contrasting management using data from two different survey methods. Results showed that overall community composition had remained stable but that change had occurred in the different management types. More detailed investigations were carried out on 14 sites which had been managed consistently as hay meadows. The meadow community had been maintained over 25 years but there were losses and gains of important species. To investigate whether the changes in meadow plant populations were linked to the fragmented distribution of the sites an analysis of the genetic diversity within sites and gene flow between sites was carried out. Microsatellite markers were used to study the population genetics of Rhinanthus minor (Yellow Rattle), a key meadow species. It was found that there were moderate levels of genetic diversity and evidence of gene flow between the meadows, and between the meadows and intermediate grasslands including road verges. However, there were also local patterns of 5 differentiation. A comparison was made with samples collected from meadows in Worcestershire where there were similar levels of genetic diversity but less gene flow. These findings suggest that meadow management should be continued but should also include the wider landscape by creating or restoring other grasslands which can function as part of a network of sites.

This thesis describes the results of a series of plant tissue culture, chemical and molecular based experiments aimed at developing an in vitro system to study the fundamental changes in chemical composition or activation of specific chemical pathways which take place during the onset and production of agarwood in Aquilaria malaccensis. Cell suspension cultures were established using callus initiated from shoot and leaf segments excised from in vitro grown plantlet of Aquilaria malaccensis. Callus was successfully established and maintained by culturing leaf segments on MS medium supplemented with 3% sucrose, 0.3% phytagel, 2.2µM of 2,4-D and 2.3 µM of BAP, and cultured under ambient culture condition i.e. 28 ± 2 ºC under continuous dark conditions. Cell suspensions were initiated from the callus lines using the same medium composition, but without the gelling agent and placed on a rotary shaker at 75 rpm. Leaf-derived callus (CS 11) was identified as the preferred source of callus due to the formation of a more homogenous cell suspension cultures which maintained continuous growth after many rounds of sub-culturing. Cell line CS 11 was used for further studies, i.e. determining the effect of elicitation on cell growth, biochemical change and gene expression. In order to effectively study the biochemical changes (sesquiterpenes and chromones production) within the cells in cultures, it would first be necessary to devise suitable analytical methods which would enable the analysis of the effect of elicitation, and to study the chemical profile of each cell culture lines. Various analytical techniques were evaluated using agarwood oil extracts (as standards) and cell cultures as target material. Solid phase micro extraction (SPME) was found to be the most effective technique in detecting the presence of sesquiterpenes and chromones within the cells in cultures. Four sesquiterpenes (alpha humulene, delta guaiene, beta caryophyllene, alpha guaiene) and four chromones (6-methoxy-2-(2-phenylethyl)- chromone, 5,8-dihydroxy-2-(2-(4-methoxyphenylethyl)-chromone, 7-hydroxy-2-(2-phenyl ethyl] chromone and 6-methoxy-2-[2-(3-methoxyphenyl)ethyl] chromones) were found to be produced in unstressed cell suspensions. However it was important to note that although chromones were detected there was no consistent production of any chromones in cell cultures. Overall, the production of sesquiterpenes in cell suspension cultures was found to be higher following elicitation using methyl jasmonate, salicylic acid and ethanol. While salicylic acid was found to enhance cell growth, methyl jasmonate was found to suppress the growth of cells. Unexpectedly the addition of alcohol (0.17µM), the solvent used to dissolve methyl jasmonate was found to have an effect on the production of sesquiterpenes specifically when applied separately where, it was found to induce higher concentration of alpha guaiene and alpha humulene as compared to methyl jasmonate or salicylic acid treatments. The correlation of increase in the production of sesquiterpenes in relation to sesquiterpene synthase expression was also explored in a preliminary study done using the ACL 154 primers whereby the increase in alpha humulene production was found to correlate with an increase in delta guaiene synthase activity suggesting that delta guaiene synthase may be responsible for alpha humulene production in Aquilaria malaccensis. In summary, the combined results of the above studies led to the development of a series of analytical methods and the establishment of an in vitro model system for Aquilaria malaccensis using cell cultures. This represents the first study successfully examining the simulated effect of artificially induced wound (elicitation), in terms of its direct influence on sesquiterpenes profile expressed, and an insight to gene expression patterns which take place within cell cultures of Aquilaria malaccensis.

Regulation of higher plant growth and development involves the control of cell growth and division, since plant cells are immobile. A key point of plant cell cycle control is the G1 to S transition, which is promoted by CyclinD/CDK complexes. Several subgroups of D-type cyclins exist in higher plants, and the genes encoding these proteins appear to be under environmental and developmental regulation. In Arabidopsis, the CYCD3 subgroup consists of three members. The roles that these genes play in growth and development are explored, and the interaction between these genes and other factors controlling plant growth and development are investigated. A role for CYCD3;1 and its putative regulator ANT in root auxiliary meristem development is shown. However, whilst ant and cycd3;1 mutants shared some phenotypes, such as increased petal cell size, reduced leaf cell number and reduced root thickness, double mutants exhibited additive phenotypes, suggesting that there is not a strong regulation of CYCD3;1 by ANT. Supporting this, a physical interaction between ANT and a putative ANT-binding site from the CYCD3;1 promoter was not detected, and evidence of CYCD3;1 transcription regulation by ANT was weak. Supporting an alternative hypothesis, evidence of coregulation of ANT and CYCD3;1 by cytokinins in roots is provided. The expression of these genes in roots required cytokinins and appeared to be correlated. Roles for all three CYCD3s and the ERECTA (ER) kinase in the regulation of primary vascular tissue development are described, and genetic evidence of a link between CYCD3s and ER is provided. These genes appear to be required for cell division events in the procambium lineage. Furthermore, ER was also found to regulate secondary growth. Thus five novel regulators of root development have been identified, and important knowledge regarding mechanisms of lateral aerial organ size control has been gained.

Aquaporins are membrane channels transporting water and small molecules across the membranes of various intracellular compartments. Plant aquaporins constitute the Major Intrinsic Protein (MIP) family, consisting of 35 members in Arabidopsis thaliana, all exhibiting expression patterns which are specific in tissue type and developmental timing. Aquaporins are thought to be vital to plant growth, development, and response to drought. However, little work has been done to clarify the exact roles of particular isoforms in each of these aspects. The MIP isoforms TIP3;1 and TIP3;2 are uniquely localised to both the plasma membrane and tonoplast. In addition, they are the only aquaporins present in the maturing and germinating Arabidopsis seeds. Their potential importance in these developmental processes has therefore been speculated, but is yet to be fully elucidated. In addition, it is suspected that a C-terminal domain unique to TIP3 isoforms is responsible for their plasma membrane localisation. However little has been done to dissect the cellular sorting route they take, or the order in which they reach both membranes. By misexpressing MIP isoforms, including TIP3, in transgenic Arabidopsis and systematically assessing the resulting effect on germination and seedling growth in water limiting conditions, it was found that increased expression of Arabidopsis thaliana aquaporin isoforms at atypical developmental time points can confer drought tolerance in both germination and seedling growth. More specifically, increasing the number of aquaporins at the plasma membrane can enhance tolerance to drought during germination, implying a fundamental role for the dual localisation of TIP3 at this developmental stage. Fluorescent protein fusions to TIP3 were employed to dissect the order of this isoform’s trafficking, and pharmacological techniques confirmed the route. From this, an inducible expression system for TIP3 has been established to study dual sorting, and TIP3 were confirmed to traffic independently of the Golgi complex in embryonic tissues, regardless of the presence or absence of their C-terminal motif.

The biosynthesis of the plant hormones strigolactone and abscisic acid is, in part, controlled by a family of enzymes known as the carotenoid cleavage dioxygenases (CCDs), which perform an oxidative cleavage reaction on a carotenoid substrate (9’-cis-neoxanthin for abscisic acid and 9-cis-β-carotene and 9-cis-β-apo-10’-carotenal for strigolactone) to form apocarotenoids, which are metabolised to the functional phytohormone. Phenotypic effects on seed dormancy and shoot branching have been observed in Arabidopsis thaliana and Zea mays on the application of a selection of hydroxamic acids based inhibitors, designed to inhibit CCDs, whereby application of the inhibitor to the plant result in a decrease in the time take for germination (abscisic acid mediated) or an increase in the number of lateral shoot branches (strigolactone mediated). However, the biochemical basis of these phenotypes is not understood. In the present thesis, carotenoid cleavage dioxygenases from the abscisic acid biosynthesis pathway (9’-cis-epoxycarotenoid cleavage dioxygenases (NCED)) and strigolactone biosynthesis pathway (CCD7 and CCD8) were produced in vitro and assayed for inhibition against the hydroxamic acid inhibitors. The results show that Z. mays NCED is indeed inhibited by the hydroxamic acid inhibitors (D2: greater than 95% inhibition at 100 μM) in a time dependent fashion, indicating that inhibition of NCED is the basis of the seed germination phenotype. On the strigolactone biosynthesis pathway, recombinant A. thaliana CCD7 is not inhibited by the hydroxamic acids. However, recombinant A. thaliana CCD8 is inhibited by hydroxamic acids that show shoot branching phenotypes (D6 53% inhibition at 10 μM), suggesting that inhibition of CCD8 is the basis of the shoot branching phenotype. Structure activity relationships have also been performed to identify the key features of the hydroxamic acids required to inhibit each enzyme. The biochemistry of several CCDs has also been investigated, along with that of the enzyme Dwarf27, an isomerase enzyme required for the isomerisation of all-trans-β-carotene to 9-cis-β-carotene on the strigolactone biosynthesis pathway. Investigations indicate that Dwarf27 from Oryza sativa could be a novel iron-sulfur protein which isomerises β-carotene via a one electron transfer to or from the β-carotene substrate. D27 is also inhibited to some extent by certain hydroxamic acids (e.g. 41% by D30 at 100 μM). Biochemical characterisation of A. thaliana CCD8 provides evidence for a two-step mechanism involving acid-base catalysis, and evidence is obtained for an active site cysteine residue. A possible mechanism for the double oxidative cleavage reaction catalysed by CCD8 is proposed. These results provide an insight into the biochemistry of the biosynthesis of the phytohormones abscisic acid and strigolactone and demonstrate that enzymes on the biosynthesis pathways of these hormones can be selectively inhibited using a chemical genetics approach. This has potential to aid the development of novel agrochemical compounds which could influence these processes to improve plant architecture and crop yield.

The rhizosphere consists of plant roots and the adjoining soil, which contains a functionally and genetically rich fungal community. The obligate plant symbionts, the mycorrhizal fungi, have been shown to receive substantial quantities of plant-derived C and play an important role in belowground C dynamics. The flux and residence time of C is however likely to be highly species-specific for rhizosphere fungi, and therefore their abundance and composition will likely have important implications on C storage belowground. Rhizosphere fungal community formation is extremely complex and despite being an area of intense research, current understanding is limited. The composition and abundance of rhizosphere fungi have been shown to vary with temporal and environmental parameters, and potentially geographical separation. However, no studies to date have analysed these parameters simultaneously to isolate the independent effects of each. Clone libraries in conjunction with TRFLP were performed before progressing to 454-pyrosequencing to profile the rhizosphere fungal community of a short rotation coppice (SRC) willow filed site. In this work, distinct seasonal fungal assemblages were shown, with N availability having a large effect in summer and geographical distance effects in autumn sampling points. Additionally, a rare large transition in the composition of the rhizosphere fungi was also demonstrated, which was most likely driven by extreme rainfall earlier in the growing season of the year of transition. Finally, using ₁₃C-labelled-CO₂ the belowground movement of recently derived photo-assimilates was shown to differ between Miscanthus x giganteus and SRC willow, however no significant fluxes were associated with rhizosphere fungal pathways in either crop. Results from this work demonstrate that some of the considerable complexity of microbial communities could have between overlooked in previous community analyses, whilst the flow of C within through mycorrhizal pathways maybe less important in bioenergy cropping systems compared to other ecosystems.

Post-translational modifications are an essential process in all levels of eukaryotic life. Some of these post-translational modifications, such as Ubiquitination, have been researched in great detail whereas the process of SUMOylation was only discovered in the late 1990’s and is less well understood. The DELLA repressors of plant elongation growth have been shown to undergo modification with both Ubiquitin and SUMO, and both the SUMOylation process and DELLA proteins have been linked to responses to abiotic stress in plants. In this project, putative SUMO sites on DELLA proteins RGA and GAI have been mutated to create transgenic lines in which each DELLA becomes non-SUMOylatable. While results for RGA overexpressing lines may require some further investigation, the results for GAIK versus GAIK/R have proven very interesting. With reduced levels of SUMOylation (GAIK/R), there is less restriction of growth in terms of bolting phenotype. In addition, SUMOylation of GAI was demonstrated to have an important role in abiotic stress responses, in terms of primary root growth in Arabidopsis thaliana. Binding data indicates interaction of SUMO with both RGA and GID1a, and although these interactions do not seem to be dependent on GA, they may have influence in preventing degradation of DELLA proteins. Analysis of OTS SUMO protease mutants demonstrates that SUMOylation and deSUMOylation through the OTS protease are necessary for the seed germination process in Arabidopsis, although only at moderate levels. The OTS protease is also demonstrated to have involvement in the seedling establishment process. Overall, this data further reinforces the assertion that SUMOylation is an essential process in plants, and that SUMOylation of DELLA proteins, and many other proteins, is integral to their response to abiotic stress.

The main aim of the thesis is to explore the interaction between pattern and process in vegetation ecology using a variety of mathematical and statistical methods. Of particular interest is what information about the dynamics of the underlying system can be gained through a single spatial snapshot, such as an aerial photograph or satellite image. The hypotheses are related to seagrass ecology, whose growth is primarily clonal and broadly exists as a monoculture and thus makes it an ideal candidate to study these interactions. The thesis broadly concerns two forms of spatial pattern and the underlying dynamics that give rise to them. The first concerns regular pattern formation, where the pattern has a characteristic length scale. Examples are abundant in natural systems, such as mussel beds, semi-arid ecosystems as well as seagrass. The developments concerned with regular pattern formation include methods of detection in a large spatial dataset, a novel stochastic model of vegetation that produces regular pattern with plausible mechanisms, the development of a new methodology to fit regular spatial pattern data to the model and the impact as well as evolutionary mechanisms of regular patterning in the presence of disease. The second form of spatial pattern exhibited in a wide variety of sessile species is scale-free or fractal patterning. Certain scaling heuristics, such as the boundary dimension of a vegetation cluster or the power-law exponent of the patch-size distribution have been used to infer properties of the dynamics. We explore these heuristics using a variety of plausible models of vegetation growth and find the circumstances under which there is a clear relationship between the spatial heuristics and the dynamics. These are then supplemented by viewing vegetation growth as an aggregation process. A novel model of vegetation aggregation with death is produced to find the origin of the ubiquitous power-law patch-size distribution found in nature. Finally the impact of scaling on the spread of disease is explored.

Long and short-range signaling systems in plants have previously been attributed to plant hormones. In animals, peptide signaling is the major mechanism in cell-cell communication, although little is known about this system in plants. The Cysteine-Rich Peptides (CRP) forms an emerging class of potential signaling ligands, which have been implicated in several signaling events during plant reproduction. However, little is known about their role during seed development. Recent studies have identified a significant number of CRP encoding genes in Arabidopsis thaliana of unknown function. A sub-class of MEG1-related CRPs were identified in seeds. These peptides contain 8 and 12 conserved cysteine residues, and undergo a rapid turnover by protein ubiquitination. A subgroup of these genes (AtMEG13/14/15) are expressed in the central cell and in the embryo-surrounding region of the endosperm during early seed development. Functional analysis of these peptides revealed seed abnormalities associated with defects in suspensor development and embryo patterning. Genetic analyses revealed that these genes are required before and after fertilization for correct embryo development. In addition, they also act in a parallel pathway to the SHORT SUSPENSOR to regulate early embryo development through the YODA mitogen-activated protein kinase signaling cascade. My findings highlight the novel existence of extra-embryonic factors that regulate early embryo development in plants.

Senescence is the final developmental stage of a plant leaf, which occurs as the organ reaches the end of its natural lifecycle. In this process, photosynthetic machinery is broken down and recycled as nutrients for use in the rest of the plant. This allows the recovery of the resources invested in the plant leaves as the plant transitions to a reproductive state. While a developmentally controlled process that occurs naturally as the organ ages, leaf senescence can also be induced prematurely by environmental stresses. The onset of senescence facilitates survival of the plant as a whole at the cost of leaf tissue. As such, senescence is controlled by regulatory systems that cross link developmental and stress response signals. One such signalling mechanism is the action of transcription factors. The NAC family transcription factor ANAC092 has been shown to promote the onset and progression of senescence during developmental and stress conditions. Timeseries gene expression data and network inference indicated ANAC092 as being a central hub in the stress response to Botrytis cinerea infection. ANAC092 was shown to be a functional gene during Botrytis cinerea infection, promoting the spread of lesions through the onset of a senescence like process. As such, ANAC092 represents a major cross-link between developmental and pathogenic stress response signals. A number of transcription factors bound to the ANAC092 promoter region in a yeast 1-hybrid experiment, including members of the NAC, MYB, ERF and AtHB transcription factor families. Interestingly, members of the same transcription factor family co-localised to regions of the ANAC092 promoter, suggesting they recognised similar sequences. Gene expression profiles and network inference were used to generate a model of regulation by these transcription factors. It was predicted certain transcription factors would regulate ANAC092 expression during specific conditions. In particular, different members of the NAC family of transcription factors appeared to regulate ANAC092 expression during Botrytis cinerea infection or developmental senescence. These predictions were tested using transgenic Arabidopsis. The NAC transcription factor recognition sequence is known for a limited subset of NAC transcription factors. In an attempt to define the binding motif for a higher proportion of the NACs, a yeast 1-hybrid library of 94 NAC transcription factors was constructed and screened against a range of Arabidopsis promoter regions. Binding motifs were reverse engineered and tested for NAC protein binding.