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The effects of a non-lethal freezing stress on chlorophyll content, moisture level and distribution, and abscisic acid (ABA) levels were examined in siliques and seeds of Brassica napus (canola). A non-lethal freezing stress resulted in the retention of chlorophyll in seed at harvest that was most pronounced for seeds 28, 32 and 36 days after flowering (DAF). This increase was primarily due to an increased retention of chlorophyll a relative to chlorophyll b. Chlorophyll retention in seeds exposed to a non-lethal freezing stress correlated with an increased ABA catabolism, as measured 1, 3 or 7 days after the stress treatment. Although the non-lethal freezing stress had no significant effect on moisture content in seeds of siliques stressed at 28-44 DAF, moisture distribution, as viewed by magnetic resonance imaging, showed an uneven drying of 32 and 40 DAF siliques after exposure to the non-lethal freezing stress. Moisture was initially lost more rapidly from the silique wall between seeds, than in control non-stressed siliques. Increased moisture loss was not due to structural changes in the vasculature of the silique/seed of stressed tissues. These results are consistent with the hypothesis that a non-lethal freezing stress-induced decrease in ABA level, during seed maturation, effects an inhibition of normal chlorophyll a catabolism resulting in mature but green B. napus seed.

Dry or fully imbibed seeds of western white pine (Pinus monticola Dougl. ex D. Don) were studied using high-resolution magnetic resonance imaging (MRI). Analyses of the dry seed revealed many of the gross anatomical features of seed structure. Furthermore, the non-invasive nature of MRI allowed for a study of the dynamics of water and oil distribution during in situ imbibition of a single seed with time-lapse chemical shift selective MRI. During soaking of the dry seed, water penetrated through the seed coat and megagametophyte. The cotyledons of the embryo (located in the chalazal end of the seed) were the first to show hydration followed by the hypocotyl and later the radicle. After penetrating the seed coat, water in the micropylar end of the seed likely also contributed to further hydration of the embryo; however, the micropyle itself did not appear to be a site for water entry into the seed. A model that describes the kinetics of the earlier stages of imbibition is proposed. Non-viable pine seeds captured with MRI displayed atypical imbibition kinetics and were distinguished by their rapid and uncontrolled water uptake. The potential of MR microimaging for detailed studies of water uptake and distribution during the soaking, moist chilling ("stratification"), and germination of conifer seeds is discussed.

Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7'-hydroxy ABA (7'OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC-tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7'OH ABA and ABA-GE also accumulated during moist chilling; however, 7'OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7'OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.

Aminoalcoholphosphotransferases (AAPT, EC 2.7.8.1 and EC 2.7.8.2) catalyze the transfer of CDP-aminoalcohols to sn-1, 2 diacylglycerol (DAG) to form phosphatidylaminoalcohols with the release of CMP. The Brassica napus L. AAPT1 gene (designated BnAAPT1) was identified from cDNA libraries of seedlings and developing seeds. Functional characterization was accomplished by heterologous expression of BnAAPT1 in a yeast strain deficient in AAPT activities. BnAAPT1 exhibited a greater preference for utilizing CDP-choline as a substrate with Vmax of 35 [14C]phosphatidylcholine nmol h(-1) mg(-1) protein and apparent Km of 32 microM while CDP-ethanolamine had a Vmax of 13 [14C]phosphatidylethanolamine nmol h(-1) mg(-1)protein and an apparent Km of 127 microM. The enzyme was activated by Mg2+, Mn2+ and phospholipid mixtures, and inhibited by Ca2+. A CDP-alcohol phosphotransferase motif, Asp99-Gly100-(X2)-Ala103-Arg104-(X8)-Gly113-(X3)-Asp117-(X3)-Asp121, was completely conserved in BnAAPT1 and its catalytic role was confirmed by scanning alanine mutagenesis. Over-expression of BnAAPT1 under the control of the double 35S promoter in transgenic Arabidopsis thaliana (L.) Heynh. plants led to elevated levels of the corresponding transcript and enzyme activity. In four of the high over-expression transgenic lines, phospholipid and fatty acid composition analyses revealed that chloroplastidic and extrachloroplastidic membranes isolated from transgenic leaves had about a 25% increase in phosphatidylcholine and in the proportions of polyunsaturated fatty acids [18:2+18:3], relative to the control. There were also consistent, but small differences observed in the proportions of 18:3 in transgenic green siliques and in 20:1 in mature transgenic seeds of these lines. Induction of Arabidopsis AAPT transcription in response to (+)-abscisic acid and low-temperature treatments, and the cold tolerance in BnAAPT1 transgenic seedlings implies that AAPT may play a role in resistance to damage at low growth temperatures.