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Forage growth in the subarctic is sub-optimal due to low soil nutrient levels. Forage crops in the Yukon Territory consistently require nitrogen (N) and phosphorus fertilization to meet plant requirements. Fertilization is expensive due to transportation costs and potentially harmful to the environment so alternative, more sustainable, sources of nutrients are being sought. Alfalfa is an alternative, but there is limited knowledge in the Yukon of the benefits and management of this crop as a replacement for fertilizer N. Experiments were carried out in south central Yukon during the 2005 and 2006 field seasons to examine the potential of co-inoculation of alfalfa with N-fixing Ensifer meliloti and phosphate-solubilizing Penicillium bilaii to increase the dry matter yield and N fixation of monoculture alfalfa (Medicago sativa) cv Peace and binary mixed alfalfa with smooth bromegrass (Bromus inermis) cv Carlton or timothy (Phleum pratense) cv Climax forage swards. Interactions between alfalfa inoculation and N fertilization and late season harvest treatments were assessed. The TagTeam® inoculant from Philom Bios was used as the rhizobium source which contains both Ensifer meliloti isolate NRG-34 and Penicillium bilaii isolate PB-50. Nitrogen fixation was determined using the total plant N difference method. Alfalfa growth and nodulation was successful in the trials. Inoculation had a positive impact on N fixation, whereas urea fertilizer at 25 kg N/ha had a negative impact in most cases. In the mixed alfalfa and smooth bromegrass stand there was a positive contribution from the alfalfa in both the establishment and second year with N fixation rates of up to 14 kg/ha. In the mixed timothy and alfalfa stand the N fixation reached 35 kg/ha in the establishment year and 102 kg/ha in the second year. In the establishment year the dry matter yield and N fixation of the TagTeam® inoculated, monoculture alfalfa plots were 3.1 t/ha and 77 kg N/ha. In the second year, the unharvested inoculated alfalfa treatment yielded 3.4 t/ha with N fixation of 66 kg/ha compared to the late harvest treatment which yielded only 1.5 t/ha and an N fixation rate of 20 kg/ha. The effects of the late season harvest are startling and reflect the importance of removing grazing animals during the fall to allow plant energy reserves to accumulate in the roots. Fertilizer N replacement is possible with the seeding of alfalfa into existing hay stands or in monoculture.

The mechanisms of plant response to nutrient limitation and utilisation are of great interest for agricultural purposes. Phosphate is a non-renewable resource and is one of the most important nutrients required for plant growth. Recently a new family of plant protein kinases, composed of 10 members, were discovered because of their involvement in stresses and their responses to the hormone abscisic acid (ABA). In Arabidopsis, all of these SnRK2 protein kinases have been shown to be activated by drought or hyperosmostic stress, with the exception of SnRK2.9. Five members are also activated by ABA treatment. Recently SnRK2.8 was linked to metabolic processes by being down regulated in low nutrient level conditions. In the present study, SnRK2.9 was investigated and shown to play a role in metabolic pathways, but in an opposite manner. Contrarily to SnRK2.8, transcripts level of SnRK2.9 is induced in response to phosphate, nitrogen, and sulphur deprivation. Interestingly, opposite to most phosphate-starvation inducible genes, sucrose decreases SnRK2.9's transcripts level. Transgenic plants that overexpress SnRK2.9 do not appear to be affected in terms of growth. On the other hand, overexpressing antisense SnRK2.9 or mutated snrk2.9 at residue Asp-123 by conversion to Glu (D123E), showed reduced plant growth. This phenotype was more pronounced in the absence of phosphate. A T-DNA knockout line for SnRK2.9 showed a 45% decrease in root and shoot biomass compared to wild-type Arabidopsis when grown under phosphate deprivation. Similar trends were observed when the Arabidopsis gene was introduced in Medicago sativa (alfalfa) under the control of the CaMV 35S promoter. Overexpressing D123E Atsnrk2.9 had a serious inhibitory effect on growth and the plants were no longer responsive to changes in phosphate levels. In Arabidopsis, the D123E snrk2.9 overexpressors had a 66% reduction in total seed yield when grown under +Pi conditions and a 33% reduction under -Pi conditions. These Arabidopsis transgenic lines do not share similar traits to the known phosphate metabolic mutants Pho1, Pho2, and Siz1. SnRK2.9 appears to play a key role in biomass and seed production. / Thesis (Ph.D, Biology) -- Queen's University, 2007-09-26 12:35:00.626

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Phosphoenolpyruvate carboxylase (PEPC) was examined during cold acclimation of seedlings of the freezing-tolerant cultivar (Medicago sativa ssp falcata cv Anik) and the relatively freezing-sensitive cultivar (Medicago sativa cv Trek) of alfalfa. With four days of cold acclimation, PEPC activity increased to 3.5-fold and 2-fold the control levels in Anik and Trek, respectively. This was associated with an increase in the level of a 110 kD PEPC protein and a decrease in the amount of a 120 kD PEPC polypeptide in both cultivars. The role of reversible phosphorylation in regulating PEPC activity was demonstrated by in vitro phosphorylation and dephosphorylation, which caused partial activation and deactivation of PEPC, respectively. In vivo phosphorylation experiments revealed that the 110 kD PEPC subunit is phosphorylated on serine residue(s) during cold acclimation in Anik but not in Trek. Increased PEPC activity could account for the 70% increase in the non-autotrophic or dark fixation of carbon observed in cold acclimated Anik seedlings. A possible role for dark carbon fixation in the cold-induced development of freezing tolerance is through the production of NADPH. Such a source of reducing power may be required for the repair of cold-induced damage and restoration of normal cellular functions.

Membrane proteins specific to cold acclimation were studied in alfalfa (Medicago falcata L. cv Anik) seedlings and cell cultures. They were identified following separation on polyacrylamide gels and localized as far as possible to specific membranes by fractionation on continuous sucrose gradient and analysis of marker enzyme assays. / With cold acclimation, certain membrane proteins from seedlings showed subtle changes (mainly increases) in their steady-state amount and in their net synthesis rate. Most of these changes were in proteins with molecular weights below 100 kDa and were associated with light fractions of the sucrose gradient, favoring a Golgi, endoplasmic reticulum or tonoplast location for the proteins. Preliminary work done on membrane proteins from cell cultures showed more pronounced changes with cold acclimation. Most of the changes were in proteins with molecular weights below 100 kDa and were associated with heavy fractions of the sucrose gradient. Since they are easier to harvest and to manipulate, cell cultures appear to be the better system to use in future studies.

The genes encoding malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD), and subunits of 2-oxoglutarate dehydrogenase (sucAB) constitute an operon in the order mdh-sucCDAB in Sinorhizobium meliloti. Regulation of the operon was studied using beta-galactosidase gene fusions. Expression of the operon was assayed in response to the carbon source provided, and over the growth of the culture. A promoter upstream of the mdh gene was identified, and although the promoter was active in S. meliloti it was not expressed in Escherichia coli. It was demonstrated that the role of 2-oxoglutarate dehydrogenase (OGD) is minimal in symbiosis, as nodules with no OGD activity formed nodules able to fix nitrogen. Alfalfa plants inoculated with strains of S. meliloti carrying extra-chromosomal copies of the mdh gene did not show any increase in shoot dry weight compared to plants inoculated with the wild-type strain.

A two year study was conducted to investigate the effect of intercropping corn with alfalfa or red clover with or without an initial application of the herbicide EPTC$ sp+$ (S-ethyl dipropylthiocarbamate + R-25788 antidote) on the weed yellow nutsedge (Cyperus esculentus L.). EPTC$ sp+$ applied prior to crop seeding in 1984 significantly reduced yellow nutsedge shoot growth in the first year but did not significantly reduce tuber populations. EPTC$ sp+$ significantly improved corn silage and grain yield and alfalfa establishment in the first year but did not significantly improve red clover establishment. In the second year, a trend of superior forage legume establishment and lower yellow nutsedge shoot production was observed in those treatments established the previous year with EPTC$ sp+$. Over the two year period, yellow nutsedge tuber populations in treatments established with EPTC$ sp+$ were significantly greater in the monocropped corn treatment than in any other treatment. In treatments established without a herbicide, yellow nutsedge tuber populations were also greatest in the monocropped corn treatment.

During the present studies we attempted to answer the following questions: (i) Does low temperature alter the phosphorylation level of proteins in isolated nuclei? (ii) Does the nuclear phosphoprotein population change during a prolonged exposure of seedlings to cold? (iii) Do heat-stable proteins accumulate in the nucleus during a prolonged exposure of seedlings to cold? (iv) Are the answers to the above three questions related to freezing tolerance? A possible relationship between the observed cold-induced changes in phosphoproteins and the level of freezing tolerance was explored by comparing the results of experiments conducted on two cultivars (Apica and Trek) of alfalfa (Medicago sativa L.) which markedly differ in their capacity for cold acclimation. / We show that the phosphorylation level of several nuclear proteins is subject to rapid and reversible enhancement by low temperature. Several phosphoproteins were found to be constitutively present in the nucleus of both cultivars. The cold-induced stimulation of the phosphorylation of many of these proteins was much greater in the relatively freezing tolerant cultivar Apica than in the relatively freezing sensitive cultivar Trek. Population of nuclear phosphoproteins was found to be considerably more complex in Apica than in Trek. During a prolonged exposure of the seedlings to 4$ sp circ$C, additional phosphoproteins were imported into the nucleus of Apica seedlings but not those Trek. / Some heat-stable proteins were constitutively present in the nucleus of both cultivars. However during the 4-day cold treatment, a large accumulation of several additional heat-stable proteins was observed in the tolerant, but not the sensitive, cultivar. (Abstract shortened by UMI.)