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    Fructan biosynthesis in Lolium perenne : tissue, cultivar and temperature effects on gene expression and protein accumulation profiles : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2008) Xue, Hong
    Cultivars of Lolium perenne with high concentrations of water soluble carbohydrates (WSCs) offer opportunities to mitigate greenhouse gas emissions (nitrous oxides) from grazed pastures and improve meat and milk production in livestock. Our previous studies demonstrated that fructan accumulation in the blades of high W SC grasses involves a strong gene x environment interaction. To identify the temperature effects on the expression of high sugar trait in the high sugar cultivars. we conducted a pot trial in climate chambers with temperature regimes set at10/10, 20/10 and 20/20°C (day/night), respectively. Water soluble carbohydrate concentrations, the expression of the key genes and proteins: l-SST (sucrose: sucrose l-fructosyltransferase), l-FFT (fructan: fructan l-fruclosyltransferase), 6G-FFT (fructan: fructan 6G-fructosyltransferase) and l-FEH l-fructan exohydrolases) involved in the fructan biosynthetic pathway of L. perenne were compared in blades and sheaths of three selected high sugar cultivars (P, A and H) and a common cultivar (F) grown under the three temperature regimes. We found that amongst the selected 3 high sugar cultivars, high molecular weight (HMW) WSC content was significantly higher in P and A cultivars, regardless of the temperature regimes. As expected, sheaths contained significantly higher concentrations of HMW WSCs (fructans) compared to leaf blades. The highest WSC contents in both leaf and sheath tissues accumulated at 10/10°C while the lowest accumulated at 20/20°C. Gene expression profiles demonstrated that all four genes studied were more significantly expressed in sheaths compared to blades, and the expression levels were highly correlated with fructan accumulation in this tissue. Low temperature resulted in significant up-regulation of l-SST in sheaths, but not in blades. l-FFT was highly expressed in blades of A and P cultivars. Unexpectedly. 6G-FFT was expressed more significantly in the control F cultivar. but not in the high sugar cultivar P. Protein expression profiles showed that l -SST protein accumulated to high levels in sheaths, whereas protein levels of l-FFT and l-FEH were higher in blades. l-SST protein levels in both blades and sheaths generally increased in plants grown at low temperatures, whereas l-FFT protein was not affected by low temperatures in blades and sheaths, furthermore, in both tissues there was no consistent effect observed between the different cultivars and temperature regimes on l-FEH protein levels.
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    Tissue-specific responses to water deficit in the New Zealand xerophytic tussock species Festuca novae-zelandiae : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2005) Clark, Gregory Thomas
    Festuca novae-zelandiae (Hack.) Cockayne is an endemic New Zealand perennial tussock forming grass of the family Poaceae. Morphologically F. novae-zelandiae exhibits a number of leaf adaptations associated with dehydration postponement as reflected in the climatic distribution of this species and its occurrence as a physiognomic dominant grass in semi arid short-tussock grasslands. Biochemical studies into the drought tolerance of this species have indicated the occurrence of tissue specific responses with respect to abscisic acid (ABA) and proline accumulation and protein turnover suggestive of a preferential protection of the tiller base and associated meristematic zones at the expense of lamina tissues. Further tissue specific biochemical responses to water-deficit stress in F. novae-zelandiae have been investigated. Changes in water-soluble carbohydrates (WSC) were monitoured over a 49-day dry-down period (decline in soil water content from 30% to 4%) in consecutive leaf segments comprising the leaf base (meristem region), elongation zone, the enclosed and exposed lamina, as well as basal sheath segments from the two next oldest leaves. In fully hydrated leaf tissues polymers of fructose (fructans) were the main WSC present and were mainly low molecular weight fructans of the inulin and neokestose series with the average degree of polymerization (DP) of fructan pools from 6 to 9. The highest fructan concentrations were present towards the leaf base. Fructan concentrations decreased over the course of the dry-down, although remained significantly higher in the meristem region of the tiller base with respect to any other tissue, until tissue water content fell below 45%. By day 49 of the dry-down period, the average DP of the fructan pool in tissues was from 3 to 5. Sucrose content increased in each tissue during the course of the dry-down, and was highest at the leaf base, where a concentration of 200 µmol g-1 dry weight was measured after 49 days of dry-down. The negative correlation between fructan and sucrose content, which indicates an inter-conversion dependent on tissue water content, suggests that, in this specises, fructans serve as a carbohydrate storage pool, while sucrose stabilises the meristem during extreme water deficit.