Phosphate (P) supply is one of the major determining factors to plant productivity, since the element affects the growth and the development of plants. In response to Pdeficiency treatment, plants display alterations in root system architecture caused by changes in primary root (PR) and lateral root (LR) length and LR density. In this thesis, the root growth of the agronomically important legume, white clover (Trifolium repens L.) was found to be slightly stimulated in terms of PR length, LR number and total LR length when plants were grown in a P-deficient media (0.01 mM orthophosphate; Pi) when compared with plants grown in a P-sufficient media (1.00 mM Pi) when using a hydroponic growth system. When plants are grown in a P-sufficient media, treatment with 100 nM exogenous 1- aminocyclopropane-1-carboxylic acid (ACC) and exogenous auxin (5 nM 1- naphthylacetic acid, NAA) resulted in significant increases in white clover PR length, LR number and LR length. However, when ethylene action or auxin transport were inhibited using 300 ppm 1-methylcyclopropene (1-MCP) and 100 nM 1-Nnaphthylphthalamic acid (NPA), respectively, root growth was significantly reduced which suggests roles for ethylene and auxin in mediating white clover root growth. To examine the effects of these hormones on plants grown in P-deficient media, 100 nM ACC treatment significantly enhanced the stimulatory effects of growth on Pdeficient media only, while exposure of plants to P-deficiency alone was sufficient to significantly neutralise the inhibitory effects of 1-MCP on root growth. Hence, exposure to P-deficiency is proposed to increase either ethylene biosynthesis or ethylene sensitivity in white clover roots. In contrast, for plants grown in P-deficient media, treatment with 5 nM NAA significantly abolished the stimulation of white clover root growth observed with P-deficiency so it is proposed that exposure to Pdeficiency increases either auxin biosynthesis or auxin sensitivity, but the 5nM NAA concentration used was too high to stimulate root growth. Using DR5p::GUS transgenic white clover, auxin activity was found in the root tips and root primordia. Using these plants, it is suggested that P-deficient treatment and ACC treatment influenced white clover root growth through an increase in auxin sensitivity. Overall, ethylene and auxin are found to be essential in mediating white clover root growth in P-sufficiency, and also in mediating root responses to P-deficiency through changes in terms of the biosynthesis and the sensitivity of these two hormones.

The investigation in this thesis is divided into two parts. In the first part, the expression and accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO), the enzyme which catalyses the final step of ethylene biosynthesis in higher plants, is examined during exposure of white clover (Trifolium repens L.) to a water deficit. The second part of this thesis is focused on the identification and characterisation of a water-deficit-associated ACC synthase (ACS), the enzyme which catalyses the production of ACC. In the first part, two white clover varieties with differing sensitivity to water deficit, a drought-tolerant Tienshan ecotype and a drought-sensitive Grasslands Challenge cv. Kopu II cultivar were exposed to two water deficit treatments: one cycle of water deficit (designated non-prestressed; NPS) and a water deficit, a rehydration period and then a second water deficit treatment (designated pre-stressed; PS) in the New Zealand Climate Environment Laboratory (NZCEL). Treatments were terminated when the petiole elongation rate (PER) in the first fully-expanded leaf reached zero. Water relations, growth responses, the expression of the white clover ACO genes, TR-ACO1 TR-ACO2 and TR-ACO3 and the accumulation of two of the corresponding proteins, TR-ACO1 and TR-ACO2, were then examined. The soil water content (SWC) and leaf water potential (LWP) measured in both varieties and in both water deficit treatments declined progressively. The rate of decline in SWC and LWP was slower in the Tienshan ecotype with no difference between the NPS and PS treatments. However, the LWP in the Tienshan ecotype at the point at which the PER ceased was less negative (ca. -1.4 MPa) compared to Kopu (ca. -1.7 MPa). In addition, the decline in the PER differed between NPS- and PS-treated Kopu. In the NPS-treated Kopu, the PER was maintained at a high rate when plants were exposed to SWC above 18%, but declined sharply as the SWC declined further. However, in the PS-treated Kopu, the PER declined more progressively in a similar pattern to that determined for NPS- and PS-treated Tienshan. Expression of TR-ACO1 and accumulation of TR-ACO1 was observed in the apical structure of the stolon. As the water deficit progressed, no significant alteration in TR-ACO1 expression and TR-ACO1 protein accumulation was observed in the apical structures of both the NPS- and PS-treated Tienshan ecotype suggesting some degree of protection of the meristem tissues in this more drought-tolerant variety. However, a discernable decline in expression of TR-ACO1 and accumulation of TR-ACO1 protein was observed in the NPS-treated Kopu suggesting some degree of tissue injury in this more drought-susceptible variety. However, after the pre-stress (PS) treatment, no real changes in TR-ACO1 expression and TR-ACO1 protein accumulation were observed, in common with the observations for the NPS- and PS-treated Tienshan ecotype suggesting that meristem protection may now be occurring. The results suggest further that the pre-stress treatment of the more drought-susceptible Kopu may result in a degree of acclimation to the water deficit. For the first-fully expanded leaves, expression of two transcripts, TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein was monitored as the SWC decreased. The expression of TR-ACO2 and accumulation of TR-ACO2 decreased as the water deficit progressed in both the NPS- and PS-treated Tienshan ecotype and correlated with the decrease in PER. By contrast, in the NPS-treated Kopu, TR-ACO2 expression and TR-ACO2 protein accumulation increased, but again, after a period of pre-stress, TR-ACO2 expression and TR-ACO2 accumulation decreased, in common with the Tienshan ecotype. Again, the pre-stress treatment of the drought-susceptible Kopu may result in a degree of acclimation to the water deficit such that the responses become similar to those observed in the more drought-tolerant Tienshan ecotype. However, in both NPS- and PS-treated Tienshan and Kopu there was no significant alteration in the expression of TR-ACO3 in the first fully-expanded leaf. The expression of TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein were also observed in the second fully-expanded leaves (an older tissue). Again similar patterns in the expression of TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein were observed in both NPS- and PS-treated Tienshan and Kopu. In these leaves, expression of TR-ACO2 and accumulation of TR-ACO2 protein decreased as the water deficit progressed, but expression of TR-ACO3 increased as the water deficit decreased to less than 10%. These results suggest that responses of younger tissues (apical structure; first-fully expanded leaf) maybe the critical determinant for the tolerant (or otherwise) of white clover plants to water deficit. In the second part of this thesis, four ACS genes were identified from the Tienshan ecotype exposed to water deficit and designated TR-ACS-T. Three of these were similar to previously identified TR-ACS genes from Grasslands Challenge genotype 10F while the fourth was a novel gene designated TR-ACS4-T. TR-ACS4-T is 64%, 64% and 63% homologous to TR-ACS1-T, TR-ACS2-T and TR-ACS3-T, respectively in terms of nucleotide sequence. In the GeneBank database, TR-ACS4-T shares highly homology to ACC synthase sequences from a wide range of tissues including seedlings and fruit tissues, in addition to a high homology to ACS genes induced in auxin-, wounding- and ethylene-treated tissues. The pattern of TR-ACS4-T expression observed during leaf development suggests that the gene is expressed initially in the apical structures and in the newly initiated leaves, and then again in the later mature leaves and those at the onset of senescence. Expression decreases again during senescence. TR-ACS4-T expression is not altered by water deficit, but is induced by both ethylene and NAA treatment, but the auxin-induced TR-ACS4-T is mediated by ethylene treatment.

Phosphate (P) supply is one of the major determining factors to plant productivity, since the element affects the growth and the development of plants. In response to Pdeficiency treatment, plants display alterations in root system architecture caused by changes in primary root (PR) and lateral root (LR) length and LR density. In this thesis, the root growth of the agronomically important legume, white clover (Trifolium repens L.) was found to be slightly stimulated in terms of PR length, LR number and total LR length when plants were grown in a P-deficient media (0.01 mM orthophosphate; Pi) when compared with plants grown in a P-sufficient media (1.00 mM Pi) when using a hydroponic growth system. When plants are grown in a P-sufficient media, treatment with 100 nM exogenous 1- aminocyclopropane-1-carboxylic acid (ACC) and exogenous auxin (5 nM 1- naphthylacetic acid, NAA) resulted in significant increases in white clover PR length, LR number and LR length. However, when ethylene action or auxin transport were inhibited using 300 ppm 1-methylcyclopropene (1-MCP) and 100 nM 1-Nnaphthylphthalamic acid (NPA), respectively, root growth was significantly reduced which suggests roles for ethylene and auxin in mediating white clover root growth. To examine the effects of these hormones on plants grown in P-deficient media, 100 nM ACC treatment significantly enhanced the stimulatory effects of growth on Pdeficient media only, while exposure of plants to P-deficiency alone was sufficient to significantly neutralise the inhibitory effects of 1-MCP on root growth. Hence, exposure to P-deficiency is proposed to increase either ethylene biosynthesis or ethylene sensitivity in white clover roots. In contrast, for plants grown in P-deficient media, treatment with 5 nM NAA significantly abolished the stimulation of white clover root growth observed with P-deficiency so it is proposed that exposure to Pdeficiency increases either auxin biosynthesis or auxin sensitivity, but the 5nM NAA concentration used was too high to stimulate root growth. Using DR5p::GUS transgenic white clover, auxin activity was found in the root tips and root primordia. Using these plants, it is suggested that P-deficient treatment and ACC treatment influenced white clover root growth through an increase in auxin sensitivity. Overall, ethylene and auxin are found to be essential in mediating white clover root growth in P-sufficiency, and also in mediating root responses to P-deficiency through changes in terms of the biosynthesis and the sensitivity of these two hormones.

Phosphate (P) supply is one of the major determining factors to plant productivity, since the element affects the growth and the development of plants. In response to Pdeficiency treatment, plants display alterations in root system architecture caused by changes in primary root (PR) and lateral root (LR) length and LR density. In this thesis, the root growth of the agronomically important legume, white clover (Trifolium repens L.) was found to be slightly stimulated in terms of PR length, LR number and total LR length when plants were grown in a P-deficient media (0.01 mM orthophosphate; Pi) when compared with plants grown in a P-sufficient media (1.00 mM Pi) when using a hydroponic growth system. When plants are grown in a P-sufficient media, treatment with 100 nM exogenous 1- aminocyclopropane-1-carboxylic acid (ACC) and exogenous auxin (5 nM 1- naphthylacetic acid, NAA) resulted in significant increases in white clover PR length, LR number and LR length. However, when ethylene action or auxin transport were inhibited using 300 ppm 1-methylcyclopropene (1-MCP) and 100 nM 1-Nnaphthylphthalamic acid (NPA), respectively, root growth was significantly reduced which suggests roles for ethylene and auxin in mediating white clover root growth. To examine the effects of these hormones on plants grown in P-deficient media, 100 nM ACC treatment significantly enhanced the stimulatory effects of growth on Pdeficient media only, while exposure of plants to P-deficiency alone was sufficient to significantly neutralise the inhibitory effects of 1-MCP on root growth. Hence, exposure to P-deficiency is proposed to increase either ethylene biosynthesis or ethylene sensitivity in white clover roots. In contrast, for plants grown in P-deficient media, treatment with 5 nM NAA significantly abolished the stimulation of white clover root growth observed with P-deficiency so it is proposed that exposure to Pdeficiency increases either auxin biosynthesis or auxin sensitivity, but the 5nM NAA concentration used was too high to stimulate root growth. Using DR5p::GUS transgenic white clover, auxin activity was found in the root tips and root primordia. Using these plants, it is suggested that P-deficient treatment and ACC treatment influenced white clover root growth through an increase in auxin sensitivity. Overall, ethylene and auxin are found to be essential in mediating white clover root growth in P-sufficiency, and also in mediating root responses to P-deficiency through changes in terms of the biosynthesis and the sensitivity of these two hormones.

The factors influencing senescence in white clover (Trifolium repens L.) are of considerable importance to the pastoral sector of New Zealand’s economy. The plant hormones, ethylene and the cytokinins, have been implicated as having opposing influences on senescence. This project focused on the cytokinins. The rate limiting step in cytokinin biosynthesis is catalysed by isopentenyl transferase (IPT) and the primary enzyme in the degradation of cytokinins is cytokinin oxidase/dehydrogenase (CKX). Both IPT and CKX genes are present as multi-gene families. A reduction in the level of active cytokinins either via a decrease in IPT expression, or an increase in CKX expression, or both, would implicate the cytokinins in developmental leaf senescence in white clover. White clover grows in a sequential pattern with leaves at all stages of development making it a good model for studying leaf development and senescence. A decrease in leaf chlorophyll is used as a marker for the onset of senescence. A micro-scale chlorophyll analysis was developed using the NanoDrop™ thus allowing tissue from the same leaflet to be used for gene expression and chlorophyll measurements. The pattern of chlorophyll changes was similar to that shown by Hunter et al.(1999) and Yoo et al.(2003) in white clover stolons used for ethylene research. Reverse transcriptase PCR (RT-PCR) and BLAST analysis was used to identify five putative IPT genes and seven putative CKX genes from white clover. RT-PCR demonstrated the expression of seven of these genes (TrIPT1. TrIPT13, TrIPT15 TrCKX1, TrCKX2, TrCKX6). Analysis with quantitative real-time PCR showed expression of TrCKX2 increased markedly during leaf expansion and was consistently high during senescence, suggesting a potential role for CKX in facilitating the progression of senescence.

Diss. (sammanfattning). Umeå : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Lic.-avh. (sammanfattning) Umeå : Sveriges lantbruksuniv. / Härtill 2 uppsatser.

Abstract Mycorrhizal fungi are important contributors to the functioning of boreal forests, since they act in the bilateral carbon and nutrient transport between above- and belowground parts of the ecosystem. In ectomycorrhizal (ECM) symbiosis of woody host plants, both fungal and plant partners depend on resources provided by the other. A single tree may simultaneously host several ECM fungal partners, which greatly enhance the host's nutrient uptake. At the same time nearly 20% of host primary production is allocated to mycorrhizal fungi. Although fungi depend on host-derived carbon, it is poorly understood how reduced carbon availability, e.g., due to herbivory, affects the ECM fungal symbionts. In this thesis I studied the impact of simulated insect defoliation or mammal browsing on mycorrhizal fungi of boreal woody hosts. Quantitative and qualitative changes in biomass partitioning in different fungal compartments were detected. None of the experiments showed that defoliation or shoot clipping treatments reduced the intensity of ECM colonisation, while treatments often shifted fungal composition towards less biomass producing ECM morphotypes. Above- and belowground diversity in ECM symbionts tended to decrease due to shoot or foliar damage. In addition, in some cases defoliation also reduced fungal biomass in fine roots and decreased ECM sexual reproduction by reducing the number of sporocarps produced. Defoliation induced a similar response pattern in the host and in ECM fungi with a stronger response to increasing severity of treatment (e.g. degree of removed foliage or repeated years of defoliation). This was also confirmed when relating the effects of host and ECM fungal symbionts to defoliation using present and previously published data. The present results suggest that belowground adaptation of boreal trees to the changing environment is mediated by changes in fungal community or biomass partitioning. The lack of response in the intensity of ECM colonisation further emphasises the importance of the symbiosis to boreal trees.

Torpedograss (Panicum repens) was first observed in Lake Okeechobee in the 1970s and appears to have displaced an estimated 6,400 ha of native plants, such as spikerush (Eleocharis cellulosa), where inundation depths are often less than 50 cm. Two series of studies evaluated substrate exposure and water depth influences on torpedograss establishment and competitiveness. Results revealed that fragments remain buoyant for extended periods and so facilitate dispersal. Once anchored to exposed substrate fragments can readily root and establish. Subsequently, torpedograss thrives when subjected to inundations to 75 cm and survives prolonged exposure to depths greater than 1 m. These findings suggest that fluctuating water levels contribute to torpedograss dispersal and colonization patterns and that low water levels increase marsh area susceptible to invasion. The competition study found that spikerush grown in monoculture produces significantly more biomass when continually inundated to shallow depths (10 to 20 cm) than when subjected to drier conditions (-25 cm) or greater inundations (80 cm). In contrast, torpedograss establishes more readily on exposed substrate (-25 to 0 cm) compared to inundate substrates. During the first growing season biomass production increases as substrate exposure interval increases. However, during the second year, established torpedograss produces more biomass when grown on intermittently wet (0 cm) compared to permanently dry (-25 cm) or intermittently inundated (10 cm) substrates. No difference in production was observed between substrates permanently inundated (10 cm) and any other regime tested. During the first two years of torpedograss invasion, regardless of treatment, spikerush suppresses invasion and torpedograss had little effect on established spikerush, indicating that spikerush-dominated areas are capable of resisting torpedograss invasion. Even so, disturbances that might cause mortality of long hydroperiod species, such as spikerush, may create open gaps in the native vegetation and thus facilitate torpedograss establishment and expansion.

Crop wild relatives (CWR) are an indispensable and at the same time threatened genetic resources for plant breeding. The study uses wild species related to celery to demonstrate how genetic resources of CWRs can be actively maintained in their natural surroundings (in-situ). Genetic reserves should be designated for long term conservation of selected occurrences. The study presents the selection procedure in detail, aiming at the identification of occurrences and sites suitable for the designation of genetic reserves, the spatial model of a genetic reserve and first practical results of the project. The overall aim of the project is the establishment of a nationwide network of genetic reserves for Apium graveolens, Helosciadium repens, H. nodiflorum and H. inundatum, the four wild celery species native to Germany. Helosciadum repens (Jacq.) W.D.J.Koch is threatened by genetic erosion due to a decline in population numbers and sizes. The loss of any population is an irretrievable loss of diversity and opportunity to enhance crops in the future. Genetic reserves are one way to conserve these populations and their genetic potential. Twenty-seven populations were selected for the analysis in a decision process based on site information. Microsatellites (SSR) were used to elucidate the genetic diversity of German populations. A cluster analysis was performed to see if the individuals form clusters of similarity. For that, a discriminate analysis of principal components (DAPC) was conducted, as the inbreeding index indicated a high number of inbreeding events in the populations and thus discordance with HWE (Hardy-Weinberg equilibrium). The analysis identified six genetic groups, which coincide well with the geographic origin of the analysed plants. The allelic richness (mean counts of alleles per individual per population) was higher in the southern populations compared to the northern ones. This North-South discrepancy was also visible as a high heterogeneity in the cluster assignments in the DAPC analysis. These differences in genetic diversity might be a result of the biogeographic history of Europe, especially the last glacial maximum. For the establishment of genetic reserves, two populations were considered as most important: The population that differs the most from the average genetic composition and the population that represents the average genetic composition of a population the best. The two extremes of differentiation were interpreted as such that the former has a specific adaptation to its local environment, and the latter represents all populations the best. DifferInt was used to analyse the SSR data and validate the differentiation of all populations compared to a pool of populations. However, SSRs are not capable of detecting adaptive traits. Populations were additionally chosen from different eco-geographic units (EGU), to increase the chance of capturing different traits. EGUs (Naturräume) are areas of specific abiotic and biotic features. These features may influence selection pressures and induce local adaptations. Based on site parameters and genetic data, 14 most appropriate wild populations (MAWP) were identified for genetic reserves establishment. For H. repens, two eco-forms are known and described in the literature. Besides their different habitats (terrestrial/semi-terrestrial and aquatic) they can be differentiated by morphological traits. Leave and stolon sizes and flowering behaviour differ significantly. Furthermore, the roots of the aquatic forms do not anchor in soil but on other aquatic plants, wood or roots of trees, while the terrestrial form exhibits a shallow root system network similar to other perennial species. To this end, no genetic analysis was conducted to clarify the phylogenetic status of the putative forms and authors avoided the usage of any specific noun rather than form. The SSR data from the previous study was evaluated, particularly with regards to the two forms. Additionally, an ISSR analysis was conducted, and the data was used to perform a PCA. There was no genetic clustering regarding the two forms neither in the SSR nor in the ISSR data. Nonetheless, the North-South discrepancy in the genetic diversity that was visible in the DAPC plot was confirmed in the PCA of the ISSR data. However, markers may fail to detect quantitative variation for adaptively important traits. As the most obvious difference in the two habitats was the water availability, the adaptation of both forms to drought stress was studied by measuring the relative water content of leaves, system water content and water loss during drought stress conditions. The stomatal index was measured for different water treatment levels. The results indicate that phenotypic plasticity rather than genotypic adaptation is responsible for different H. repens phenotypes.