This thesis reports the development of two rapid thermal annealing systems, one based on resistive heating of graphite strips, the second on heating from incoherent lamp radiation. Electrical activation studies of silicon implanted gallium arsenide has been used to compare the systems with those available commercially. It has been shown that commercial systems can yield temperature measurement errors in excess of 50° C. Furthermore, the systems have been used to investigate the electrical activation of silicon implants co-implanted with other ions into gallium arsenide, with a view to either, improving the activation of the silicon for high doses, or modifying the carrier profile shape for low doses. A factor of two improvement in the electrical activation of high dose silicon implants has been achieved by the co-implantation of phosphorus, with a reduction in the annealing temperature required to achieve a given activity also being observed. An alternative processing methodology is also proposed for through- nitride implantation. Phosphorus implants have also been used to "pre-amorphise" substrates to prevent ion channelling. Providing the damage is maintained below a certain level, improvements in profile shape can be obtained. Other compensation techniques using boron and carbon implants have also been investigated. Boron has been demonstrated to provide improved carrier activation for low implant doses, with thermally stable profile modification capability as the dose is increased. The electrical activation of single carbon implants (40% maximum) is below the level of compensation of silicon implants (approximately 90%) co-implanted with carbon. This in turn means carbon is excellent for profile modification as no p-type layer is created beyond the donor implant.

Nitrate assimilation is an extremely important part of the nitrogen cycle and is carried out by most bacteria, fungi and plants. A relatively short catalytic pathway reduces nitrate to nitrite (via nitrate reductase activity) and then nitrite to ammonium ions (via nitrite reductase activity) which are converted into organic nitrogen by further metabolic pathways. A considerable amount of information is known about the biochemistry, genetics and recently, the molecular biology of these two enzymes. Much less is known about the transport of nitrate and nitrite into cells as well as the synthesis of the molybdenum cofactor needed for nitrate reductase catalytic activity. Research work reported in this thesis focus on these latter two processes in the eukaryotic model organisms. Aspergillus nidulans and to a lesser extent Arahidopsis thaliana. Genetic characterisation of 47 crn mutants shows that there are three additional genes (i.e. to the original identified crnA gene) likely to be involved in nitrate transport. These additional genes are unlinked to each other or to crnA. Although it was shown that the nitrate uptake into cells of these mutants are lower than the wild-type, their exact involvement in nitrate transport requires their molecular cloning. Certain mutations generated in the crnA gene have been investigated at the molecular level and the disruptions in the protein determined. During the genetic studies of crn mutants, two other genes were postulated. The first is chlA, mutation which results in resistance to chlorate (unlike the wild-type) and caesium (like the wild-type). The second is cesA mutation. These latter mutants lead to caesium sensitivity but are chlorate sensitive like the wild-type. These two genes are unlinked to crnA, crnB, crnC and crnD genes. The bases of these phenotypes is unclear and need further investigation. A study of nitrite uptake was undertaken which showed that wild-type A. nidulans has an active nitrite transport system. The activity of this system is repressed by ammonium and is nitrate induced. Mutants which are hypersensitive to chlorate taken up much higher levels of nitrite as compared to wild-type. 2,082 cnx mutants were isolated and 456 of these were classified as cnxA, cnxB, cnxC, cnxE, cnxF, cnxG and cnxH mutants on the basis of phenotypic complementation. No novel cnx genes were found. More importantly a number of temperature -conditional mutants were isolated, 10 mutants were found to be temperature-sensitives and 10 cryo-sensitives. Of the isolated temperature-sensitives 1 located in cnxA, 1 in cnxB, 2 in cnxC, 1 cnxE, 2 in cnxF and 3 in cnxH. Of the crysosensitives 4 in cnxB, 3 in cnxC and 3 in cnxF. These mutants will be particularly useful to relate structure and function when data is forthcoming regarding their protein sequence. Two temperature-sensitive mutants, cnxH255 and cnxH261 showed reduced nitrate reductase thermostability which indicates that the cnxH product could be associated with the NR protein. One of the Aspergillus nidulans genes required for the synthesis of the molybdenum cofactor was isolated using molecular self-cloning transformation approaches. This gene, cnxH, was sequenced at the nucleotide level as well as three mutant alleles (one temperature sensitive and two temperature non-conditional). The results show that the cnxH product is the homologue of Escherichia, coli moaE whose role is in the synthesis of the molybdenum cofactor specifically to convert the large subunit to active converting factor. Sequence analysis of the two non-conditional mutants indicates that such mutants generated stop codons which provides little or no information about the structure / function relationships. The mutation in the temperature-sensitive mutant lead to a glycine insertion at position +443 and it is postulated that this additional amino acid caused the heat liability of the NR enzyme. Studies of cnxH expression show that the cnxH is in very low abundance and not regulated at the transcriptional level at least since similar transcript levels were seen in both nitrate and ammonium grown cells- conditions, which making difference for nitrate reductase activity. Finally attempts at isolating Arabidopsis thaliana cnx genes failed.

Leaves of tropical and sub-tropical species are rapidly chill-injured when exposed to temperatures in the 0 to 10°C range. In contrast, leaves of temperate and arctic alpine species can withstand these temperatures without damage. Chill-sensitive leaves can be divided into two categories based on their susceptibility to chilling-injury:- 1) Extremely chill-sensitive species which are rapidly damaged on exposure to temperatures between 12 to 15°C (e. g. Episcia reptans) and which cannot be readily hardened against chilling-injury. Maintaining 100 per cent relative humidity during chilling at 5°C does not prevent injury to these species, although the speed at which injury occurs is reduced. 2) Chill-sensitive species which are damaged in the 0 to 10°C range (e. g. Phaseolus vulgaris) and which can be readily hardened against chilling-injury at 5°C, 85 per cent RH, by 4 days growth at 12°C, 95 per cent RH, before chilling. Maintaining 100 per cent RH during t chilling at 5°C prevents injury to these species. It is considered that the primary response in chilling-injury is a temperature induced phase transition in the lipids of the cellular membranes from a liquid-crystalline to a solid gel state. The temperature at which the phase change occurs appears to be determined mainly by the degree of unsaturation of the fatty-acids associated with the phospholipids. The chill-sensitivity of leaves grown at 25&°C was related to a low percentage of linoleic and linolenic acid associated with each phospholipid. The degree of unsaturation of the glycolipids could not be related to, the chill-sensitivity of the species. Hardening the chill-sensitive species Phaseolus vulgaris and Gossypium hirsutum against chilling-injury at 5°C, 85 per cent RH, resulted in increases of up to 12 per cent in the percentage of linoleic acid associated with all the leaf phospholipids. The degree of unsaturation of the glycolipids did not change during hardening. The increases in unsaturation of the phospholipids were shown to be positively related to the increased tolerance of the plants to chilling by the fact that similar increases did not occur during the growth of chill-resistant Hordeum vulgare at 12°C and the ineffective attempts at hardening chill-sensitive Episcia reptans over 4 days at 15°C, the lowest temperature this species can withstand without injury. Chilling-injury in Phaseolus vulgaris could be prevented by enclosing the plants in polythene bags at 5°C, thus maintaining 100 per cent RH. However, leaves of this species transferred from 25°C to 5°C. 100 per cent RH and 12°C, 100 per cent RH for 4 days did not harden against subsequent chilling-injury at 5°C 85 per cent RH. In agreement with this finding no increase in unsaturation of the phospholipids was detected over 4 days growth at 5°C 100 per cent RH, and 12°C 100 per cent RH. It is suggested that plants grown at 5 and 12°C 100 per cent RH, by enclosure in polythene bags do not harden against subsequent chilling- injury at 5°C 85 per cent RH, because the carbon dioxide concentration within the bag is rapidly lowered by photosynthetic fixation. This results in the cessation of photosynthesis and a reduced supply of the cofactors oxygen and NADPH available for desaturase activity, thereby preventing an increase in the degree of unsaturation of the phospholipids. An increase in leaf age at 25°C was shown to increase the susceptibility of chill-sensitive plants to chilling-injury. This increase in susceptibility of older leaves to damage was related to a decrease in the degree of unsaturation and weight of phospholipids with increase in physiological age at 25°C. In conclusion, the results reported in this investigation provide evidence that hardening chill-sensitive leaves prevents chilling-injury by increasing the degree of unsaturation of the membrane phospholipids thereby lowering the transition temperature of the lipid layer of the cellular membranes. This phase change does not occur on chilling chill-resistant plants but may occur at sub-zero temperatures and increase their susceptibility to freezing-injury.

Nitrite reduction is the third step of the nitrate assimilation pathway in higher plants and is catalysed by nitrite reductase. The whole-plant barley mutants STA1010, STA2760 and STA4169 accumulate nitrite in the leaf after treatment with nitrate and, like the nir1 mutant STA3999 (Duncanson et al, 1993), lack detectable nitrite reductase cross-reacting material in the leaf and root. STA1010, STA2760 and STA4169 carry a recessive mutation in a single nuclear gene, identified as the Nir1 locus. RFLP analysis of the nir1 mutant STA3999 has allowed the Nir1 locus to be mapped to within 0.3cM of the nitrite reductase apoprotein gene, Nii. Studies to confirm the identity of the Nir1 locus as Nii, by establishing the full-length Nii cDNA sequences from STA3999 and from its wild-type cv Tweed for comparative purposes, were unsuccessful as attempts to isolate a Nii cDNA clone from a barley cv Tweed cDNA library yielded only partial-length Nii clones. These nirl mutants display greatly reduced nitrite reductase activity and increased NADH-nitrate reductase activity in the leaf, as compared to wild-type plants, suggesting a regulatory perturbation in the expression of the Nar1 gene. Northern analysis shows that the nir1 mutants possess nitrite reductase apoprotein (nii) transcript of wild-type size (2.3kb) and at approximately wild-type levels. Since nir1 mutants possess a phenotype that might be anticipated for a Nii mutant, it is likely that the nir1 mutation is present in the nitrite reductase apoprotein gene Nii and affects translation of the nii transcript. Studies of barley wild-type cv Golden Promise have demonstrated that nitrite reductase in leaf tissue is up-regulated by a coaction of nitrate and light which acts, at least partly, at the transcriptional level.

The steps involved in plant reductive assimilation of sulphate to sulphide for incorporation into cysteine are not clear. The aim of the project described in this thesis was the isolation of genes encoding sulphate assimilation enzymes which will provide molecular tools for unravelling this key metabolic pathway. Functional complementation of the Escherichia coli cysteine auxotrophic strain JM15 (serine acetyltransferase deficient) using an Arabidopsis thaliana cDNA library in the expression vector YES resulted in the isolation of at least three members of an A. thaliana multigene family encoding serine acetyltransferase. Characterisation of one clone, Sat-1, showed that it conferred serine acetyltransferase activity (with apparent KM for substrates acetyl CoA and L-serine of 0.043 and 3.47 mM, respectively) on strain JM15. The 1515 bp full-length cDNA encodes a deduced protein of 391 amino acids, SAT-1, that has significant identity with bacterial and plant serine acetyltransferases, and that contains a putative N-terminal organellar targeting peptide. Southern hybridisation indicated that Sat-1 is present as a single copy in A. thaliana, while northern analysis revealed a single message of 1.5 kb. Using the A. thaliana cDNA library in the expression vector YES, cDNAs encoding a novel putative "APS reductase" were obtained by functional complementation of E. coli cysteine auxotrophic strains JM81A (adenosine 5'-phosphosulphate [APS] kinase deficient) and JM96 (3'-phosphoadenosine-5'-phosphosulphate [PAPS] reductase deficient). Retransformation of three clones (Papsr-19, Papsr-26 and Papsr-43) encoding different putative APS kinase isoforms into strain JM96 conferred low PAPS reductase activity on the mutant, although this activity was thioredoxin-independent unlike wild-type bacterial activity. The putative APS reductase has a PAPS reductase-like C-terminal domain, but further analysis demonstrated that the enzyme accepts APS in preference to PAPS as substrate and has a thioredoxin-like C-terminal domain. Isolation and characterisation of these genes invites a new hypothesis for plant reductive sulphate assimilation and provides direction for future research.

This thesis is mainly based on work done in Loch Kilconquhar, Fife, and also to a lesser extent in Loch Lindores, Fife, from March 1979 to March 1981. It discusses the seasonal productivity of the lakes and also the nutrient concentrations, with special reference to phosphate. The overall productivity of phytoplankton in Loch Kilconquhar is high compared with Loch Lindores. The high plankton densities in Loch Kilconquhar during winter were attributed to Diatom species such as Stephanodiscus. Anabaena flos-aquae formed the massive bloom during May 1980 and reached the maximum value of 461.84 +/- 47.02 mg m-2 chlorophyll a in the middle of the month, which was followed by a second blue-green bloom of Aphanizomenon flos-aquae in late summer. The submerged macrophytes such as Myriophyllum spicatum. Zanichellia palustris and Enteromorpha intestinalis grew well on the west side of the loch after the decline of the Anabaena bloom in July 1980, The value of sedimentary chlorophyll reached a maximum of 13.96 +/- 2.04 mg m in late April 1980. The high concentration of Soluble Reactive Phosphate (range: 0.004 - 0.780 mg/l PO4-P) and Nitrate-nitrogen (range: 0.980 - 2.350 mg/l NO3-N) also indicates that Loch Kilconguhar is a nutrient-rich freshwater loch. It is interesting to note that the soluble phosphate is exceptionally high compared with other freshwater lochs in Scotland. There are several possible reasons for this high concentration. Firstly, drainage from an agricultural area may contain much phosphate. However, the inflow in this case has little soluble phosphate but is high in soluble nitrate. Secondly, decomposition of organisms, notably phytoplankton blooms and macrophytes, may contribute to the high concentration of phosphate; these organisms, however, must in turn obtain their phosphorus from water and sediment. The third possibility, and probably the most important, is the nutrient release from the loch sediment. In a laboratory experiment, it was shown that when the dissolved oxygen dropped below 1 mg/1 and the redox potential E7 fell below 240 mVolt, substantial amounts of nutrients, particularly phosphate, were released into the overlying water. The primary source of nutrient in Loch Kilconquhar is the phosphorus-rich excrements of the large wildfowl population and also gulls on the loch. The results show that one g of fresh duck dropping has a mean content of 4170 +/- 350 mg/kg total phosphate and one g of gull dropping has 5072 +/- 748 mg/kg total phosphate.

The physiological ecology of five coastal species has been examined with respect to temperature and its effect on survival and distribution. The aims of the study have been to establish whether any direct correlation exists between distribution and the responses of the plants to temperature at different stages in the life cycle. The species Tinder consideration were the northern Ligusticum scoticum and Mertensia maritima and the southern Crithmum maritimum, Limonium binervosum and Glaucium flavum. Highest germination percentages for each species were found at temperatures close to those associated with the season favourable for germination in the natural habitat. Northern species had higher temperature requirements than the southern, corresponding to spring/ summer and autumn or spring germination respectively. Root respiration, measured as oxygen uptake, was found to be twice as great in the northern Ligusticum and Mertensia as in the southern Crithmum and Limonium over a range of experimental temperatures. This varied to some extent with time and temperature of pretreatment. The single experiment on the southern Glauci.um showed rates similar to those of the northern species. Arrhenius plots' of respiration data for the northern species showed a break in gradient at the upper end of the experimental temperature range which correlated well with apparently limiting July mean temperatures from the distribution maps. The southern Crithmum showed a break at lower temperature range close to the limiting January mean temperature. The response of Limonium to experimental temperature depended on the pretreatment; upper range breaks were shown after low pretreatment temperatures, and lower range breaks after higher pretreatment temperatures. The single experiment on Glaucium gave a straight line Arrhenius plot. Carbohydrate analyses of the same pretreated plants yielded additional information relevant to the survival and thus to the distribution in relation to temperature. The southern Crithmum had the highest starch content at all temperatures while the northern Ligusticum and Mertensia had less. Ratios of soluble sugar to starch were greatest in the northern species, possibly reflecting displacement of the equilibrium from starch to soluble sugar at lower temperatures. Overall a connection has been demonstrated between the direct effects of temperature on the plants and the limitation of distribution by temperature. This is clearest for the two northern species, Ligusticum scoticum and Mertensia maritima, less definite for the southern Crithmum maritimiim, and only suggested for Limonium binervosum with its apparently less simple temperature responses. Glaucium flavum appears anomalous and requires further study.

A comparative study of the carbohydrate metabolism of roots of pea (Pisum sativum var. meteor) and maize (Zea mays var kelvedon glory) seedlings was undertaken at low temperatures (2-14°C) with the aim of demonstrating differences between these species which may be associated with the differing growth capacities of their roots mot this temperature range. Pea roots displayed linear growth rates at all temperatures tested whereas maize roots ceased growth over five days at temperatures below 6°C At the respective temperatures which were minimal for root growth of the two species, roots behaved similarly with regards to soluble sugar content; firstly, total content was maintained in the roots at the initial level, and secondly, sucrose content was at its highest value and glucose content at its lowest. With rise in temperature sucrose content declined while glucose content increased. In maize roots kept at those temperatures where growth was not sustained this relationship broke down. Total sugar content of the roots was not maintained, glucose content was abnormally high and sucrose content very low. Similarly, respiration rate of maize roots at 2°C was abnormally low. When seedings were grown with roots bathed in an external solution of glucose at 2°C (or of glucose or of sucrose at 6°C the disturbances to sugar metabolism and respiration rate of maize roots were partially alleviated and this was associated with a greater amount of growth made by the roots. Examination of the activity and Km of acid invertase extracted from the roots and partially purified, showed that the sucrose levels in roots of both species were inversely related to invertase activity. However in pea, but not in maize roots, Km values for invertase showed a lowering in value after growth of seedlings at 2°C compared with 20°C. Furthermore, in pea, after growth of seedlings at 14 or 2°C Km determined at 2°C was significantly lower than when determined at 14°C. These properties are of adaptive significance at low temperatures since they will act to maintain an appreciable reaction rate. Shifts in Km of a homeostatic nature with respect to temperature were not recorded for invertase from maize roots and in this species the failure to control invertase activity at low temperatures with consequent depletion of sucrose may be associated with the inability of this species to show sustained growth at 2°C. Examination of the Km of MDH likewise revealed a shift in Km value tending to buffer the effect of temperature on reaction rate for MDH from pea but not from maize roots.

Following reports that limited gene transfer may be facilitated by making crosses with irradiated pollen, a debate has arisen as to the cause of the observed results. If maternal trends that occurred in the second generation (the M2) were largely due to the persistence of radiation induced damage, then pollen irradiation would be of little value to plant breeders. But if much of the paternal genome had been eliminated, the method could offer breeders a cheap and simple means of transferring just a few characters from one plant genome to another. By carrying out reciprocal irradiated and control crosses, it was shown in this study with barley that mutational damage is not widespread in the M2. However, consistent trends away from the F2 towards the maternal expression were not observed either. When instead the female gamete was irradiated, moderate shifts to the paternal expression did occur. As trends were for increased vigour, mutational damage is unlikely to have been the cause of these observations. By contrast, when irradiated pollen crosses were made between three varieties of potato, the M1 and M2 were consistently lower scoring than the controls. It is suggested that the results may have been different in the two species because polyploids such as the potato may be better able to tolerate radiation damage than diploids such as barley. Gamete irradiation may, therefore, be of little value in polyploid crops. It is recommended that, at least in diploid species, ovule irradiation should be further investigated because not only may it be more effective than its male equivalent, but it may also be easier to perform. Both techniques may be useful in breaking down linkages resistant to conventional crossing.

This thesis investigates physiological mechanisms underlying temperature adaptations in perennial grasses. A comparative approach was employed by using pairs of species that have been collected from lowland sites in thermally contrasting habitats up to 2000 km apart on a north-south direction. The population samples from northern latitudes, i.e. from cooler climates, had higher growth rates at 12°C than southern provenances. Dark respiration rates between 5 and 25°C did not differ between provenances; but acclimatization at 10°C increased the respiration rate in all northern population samples, whereas some southern population samples reduced their respiration rate as a response to acclimatization at 10°C. Northern provenances showed a greater activity of carbohydrate formation in light after periods of 48 hours starvation in darkness. This property could be attributed to the higher activity of RuBP-carboxylase in northern genotypes. No evidence was found that the higher activities are due to higher enzyme concentrations in these genotypes. The enzyme exhibited lower energies of activation in northern provenances in the range 5 - 15°C Activation energies for succinate dehydrogenase in isolated mitochondria were higher for northern population samples in the range 10 - 25°C. Higher specific activities were found for malate dehydrogenase from northern plants between 5 and 25°C. In this temperature range the apparent energies of activation were lower for northern plants. The enzyme displayed positive thermal modulation in both northern and southern provenances. Temperature changes had generally a greater effect on northern provenances. The significance of these findings is discussed in the light of previous reports in the literature and in relation to a possible advantage in the plant's native habitat.