Activated oxygen or oxygen free radicals have been implicated in a number of physiological disorders in plants including freezing injury. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H202 and thereby reduces the titer of activated oxygen molecules in the cell. To further examine the relationship between oxidative and freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago safiva 1.). The Mn-SOD cDNA from Nicotiana plumbaginifolia under the control of the cauliflower mosaic virus 35s promoter was introduced into alfalfa using Agrobacferium tumefaciens-mediated transformation. Two plasmid vectors, pMitSOD and pChlSOD, contained a chimeric Mn-SOD construct with a transit peptide for targeting to the mitochondria or one for targeting to the chloroplast, respectively. The putatively transgenic plants were selected for resistance to kanamycin and screened for neomycin phosphotransferase activity and the presence of an additional Mn-SOD isozyme. Detailed analysis of a set of four selected transformants indicated that some had enhanced SOD activity, increased tolerante to the diphenyl ether herbicide, acifluorfen, and increased regrowth after freezing stress. l h e F1 progeny of one line, RA3-ChlSOD-30, were analyzed by SOD isozyme activity, by polymerase chain reaction for the Mn-SOD gene, and by polymerase chain reaction for the neo gene. RA3-ChlSOD-30 had three sites of insertion of pChlSOD, but only one gave a functional Mn-SOD isozyme; the other two were apparently partia1 insertions. The progeny with a functional Mn-SOD transgene had more rapid regrowth following freezing stress than those progeny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD serves a protective role by minimizing oxygen free radical production after freezing stress.Activated oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radical are associated with a number of physiological disorders in plants. Although activated oxygen is produced as a by-product of normal cell metabolism, its levels are enhanced by exposure to chemical and environ-'

Cu/Zn superoxide dismutase (SOD) mutations are involved in about 20% of all cases of familial amyotrophic lateral sclerosis (FALS). Recently, it has been proposed that aberrant copper activity may be occurring within SOD at an alternative binding, and cysteine 111 has been identified as a potential copper ligand. Using a commercial source of human SOD isolated from erythrocytes, an anomalous absorbance at 325 nm was identified. This unusual property, which does not compromise SOD activity, had previously been shown to be consistent with a sulfhydryl modification at a cysteine residue. Here, we utilized limited trypsin proteolysis and mass spectrometry to show that the modification has a mass of 32 daltons and is located at cysteine 111. The reaction of SOD with sodium sulfide, which can react with cysteine to form a persulfide group, and with potassium cyanide, which can selectively remove persulfide bonds, confirmed the addition of a persulfide group at cysteine 111. Gel electrophoresis and glutaraldehyde cross-linking revealed that this modification makes the acid-induced denaturation of SOD fully irreversible. Furthermore, the modified protein exhibits a slower acid-induced unfolding, and is more resistant to oxidation-induced aggregation caused by copper and hydrogen peroxide. Thus, these results suggest that cysteine 111 can have a biochemical and biophysical impact on SOD, and suggest that it can interact with copper, potentially mediating the copper-induced oxidative damage of SOD. It will be of interest to study the role of cysteine 111 in the oxidative damage and aggregation of toxic SOD mutants.

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BN28 is a low-temperature-induced

We have purified to near homogeneity a recombinant form of the protein BN28 (rBN28), expressed in response to low temperature in Brassica napus plants, and we have determined its solution structure. Antibodies raised against rBN28 were used to characterize the recombinant and native proteins. Similar to many other lowtemperature-induced proteins, BN28 is extremely hydrophilic, such that it remains soluble following boiling. lmmunoblot analysis of subcellular fractions indicated that BN28 was not strongly associated with cellular membranes and was localized exclusively within the soluble fraction of the cell. Contrary to predicted secondary structure that suggested significant helical content, circular dichroism analysis revealed that rBN28 existed in aqueous solution largely as a random coil. However, the helical propensity of the protein could be demonstrated in the presence of trifluoroethanol. Nuclear magnetic resonance analysis further showed that rBN28 was in fact completely unstructured (1 00% coil) in aqueous solution. Although it had earlier been speculated that BN28-like proteins from Arabi-