No abstract

To determine whether overexpression of Fe-superoxide (SOD) dismutase would increase superoxide-scavenging capacity and thereby improve the winter survival of transgenic alfalfa (Medicago sativa L.) plants, two genotypes were transformed with the vector pEXSOD10, which contains a cDNA for Arabidopsis Fe-SOD with a chloroplast transit peptide and cauliflower mosaic virus 35S promoter. A novel Fe-SOD was detected by native PAGE in both greenhouse-and field-grown transgenic plants, but activity varied among independent transgenic plants. The increased Fe-SOD activity was associated with increased winter survival over 2 years in field trials, but not with oxidative stress tolerance as measured by resistance of leaves to methyl viologen, a superoxide generator. Total shoot dry matter production over 2 harvest years was not associated with Fe-SOD activity. There was no detectable difference in the pattern of primary freezing injury, as shown by vital staining, nor was there additional accumulation of carbohydrates in field-acclimated roots of the transgenic alfalfa plants. We did not detect any difference in growth of one transgenic plant with high Fe-SOD activity compared with a non-transgenic control. Therefore, the improvement in winter survival did not appear to be a consequence of improved oxidative stress tolerance associated with photosynthesis, nor was it a consequence of a change in primary freezing injury. We suggest that Fe-SOD overexpression reduced secondary injury symptoms and thereby enhanced recovery from stresses experienced during winter.

To test the hypothesis that enhanced tolerance of oxidative stress would improve winter survival, two clones of alfalfa (Medicago sativa) were transformed with a Mn-superoxide dismutase (Mn-SOD) targeted to the mitochondria or to the chloroplast. Although Mn-SOD activity increased in most primary transgenic plants, both cytosolic and chloroplastic forms of Cu/Zn-SOD had lower activity in the chloroplast SOD transgenic plants than in the nontransgenic plants. In a field trial at Elora, Ontario, Canada, the survival and yield of 33 primary transgenic and control plants were compared. After one winter most transgenic plants had higher survival rates than control plants, with some at 100%. Similarly, some independent transgenic plants had twice the herbage yield of the control plants. Prescreening the transgenic plants for SOD activity, vigor, or freezing tolerance in the greenhouse was not effective in identifying individual transgenic plants with improved field performance. Freezing injury to leaf blades and fibrous roots, measured by electrolyte leakage from greenhouse-grown acclimated plants, indicated that the most tolerant were only 1°C more freezing-tolerant than alfalfa clone N4. There were no differences among transgenic and control plants for tetrazolium staining of field-grown plants at any freezing temperature. Therefore, although many of the transgenic plants had higher winter survival rates and herbage yield, there was no apparent difference in primary freezing injury, and therefore, the trait is not associated with a change in the primary site of freezing injury.

Alginate, a natural polysaccharide, has been widely used in tissue engineering and drug delivery, but like other biomaterials, it causes inflammation by unknown mechanisms. We hypothesized that alginate would stimulate innate immune responses through macrophage receptors. In this study, we showed that sodium alginate induced activation of macrophage-like cells (RAW264.7) through the NF-kappaB pathway. Production of proinflammatory cytokines, such as IL-1beta, IL-6, IL-12, and TNF-alpha was time and dose-dependent. Treatment with alginate solution caused responses that closely paralleled stimulation by lipopolysaccharide in timing and magnitude. These data suggest that sodium alginate causes innate immune responses through NF-kappaB activation and likely activates the same pathways as pathogen recognition.

A physical barrier exists in Vitis vinifera L. (wine-quality grapevines) between the canes that freeze at subzero temperatures and the buds that supercool, thereby avoiding ice formation in these delicate organs. Our objective was to characterize the ice nucleation barrier by observing grape buds at different stages of acclimation, by treating them with pectinase, and by comparing them to buds of the more hardy Vitis riparia L. and to non-supercooling buds of Populus nigra L. "Italica." Differential thermal analysis (DTA) indicated that as V. vinifera andV. riparia acclimated in the autumn, the ice nucleation point of the buds declined, then increased as the grapevines de-acclimated in the spring. Laser scanning confocal microscopy was used to detect the penetration of fluorescent rhodamine green dyes from the cane into the bud as a measure of apoplastic permeability. These pore size exclusion tests indicated that the bud axes were impermeable to a 3000 MW dextran-conjugated dye when the ice nucleation temperature was below -20°C, but permeable above -20°C. This lower porosity presumably restricts the penetration of ice into the bud from the cane. In contrast to the two Vitis species, the permeability of Populus buds did not limit ice propagation, and the water in buds froze between -5 and -10°C according to DTA. Pectinase and phosphate treatments of grape buds to remove pectin increased apoplastic permeability, increased the ice nucleation temperature, and prevented supercooling according to DTA. Light microscopy indicated that suberin coated the scales of the grape buds, probably preventing ice nucleation from the environment, but was not present in the bud axis region. We concluded that a permeability barrier, possibly containing pectin, in the axis of grape buds limits the propagation of ice into the buds and enables supercooling to occur.Key words: supercooling, freezing, acclimation, winter hardiness, pectin, confocal microscopy.