The nucleotide sequence of a full-length cDNA, the deduced amino acid sequence, and the regulation of expression of a cold acclimation-specific gene, casli3, in cell suspension cultures of a freezing-tolerant cultivar of alfalfa (Medicago falcata cv Anik) have been determined. The deduced polypeptide, CAS18, i s relatively small (17.6 kD), is highly hydrophilic, is rich in glycine and threonine, and contains two distinctive repeat elements. It exhibits homology with members of the LEA/RAB/dehydrin family of proteins, which accumulate in response to abscisic acid (ABA) or water stress. It is intriguing that casl8 i s induced by neither ABA nor water stress. The casl8 cDNA hybridizes to three transcripts of 1.6, 1.4, and 1.0 kb, and the cDNA characterized here corresponds to the 1.O-kb transcript. The expression of this gene is about 30-fold greater in cold-acclimated cells than in nonacclimated cells. Although the accumulation of transcripts during cold acclimation is relatively slow, their disappearance during deacclimation i s dramatically rapid, becoming undetectable in less than 5 h. Studies of nuclear run-on transcription show that cold acclimation enhances the transcription of this gene nearly 9-fold. The stability of cas78-detectable transcripts during deacclimation is considerably increased if transcription is inhibited with cordycepin. It therefore appears that low temperature regulates the expression of casl8 at both the transcriptional and posttranscriptional levels.

Peroxidase isoenzymes may be separated on acrylamide gels and then detected by supplying the substrate in an appropriate reaction system. One such system frequently used contains guaiacol as the hydrogen donor, although this compound has certain drawbacks. Ways of circumventing these drawbacks are suggested, so that quantitative estimates of the activity of individual peroxidase isoenzymes may be obtained.

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In the F1 hybrids between Durrant's L and Durrant's S flax genotrophs, the relative mobilities of the anionic peroxidase isozymes were essentially the same as those in the L parent. The isozymes in both parents and their F1's were compared over a range of acrylamide gel concentrations, with plots of log relative mobility against gel concentration. Plots of comparative mobility, relative to the internal standard hemoglobin, against concentration were also examined. Both approaches provided evidence that apparent molecular weight modifications underlay the shift in mobility between the parents and the resemblance of the F1's to L, the parent which was homozygous for the dominant alleles controlling the mobility shift for at least two of the isozymes.

A flax (Linum usitatissimum L.) lambda gt10 cDNA library was screened with a probe coding for the amino terminus of a flax peroxidase. The probe was generated by PCR amplification of the library with one of the lambda gt10 sequencing primers and a mixed oligonucleotide derived from a well-conserved amino acid region (distal heme ligand) found in all plant peroxidases. A positive clone (FLXPER2) was isolated and characterized. The cDNA contains 1153 nucleotides, excluding the poly(A) tail, and encodes a mature protein of 297 amino acids with a molecular mass of 31.9 kDa. The mature protein's amino acid sequence contains three highly conserved regions, two of which contain histidine ligands for the heme group. The deduced amino acid sequence has nine cysteine residues. Eight are identically located to those of horseradish C peroxidase, which participate in four disulfide bridges; these cysteines are highly conserved in all plant peroxidases. One potential N-glycosylation site (Asn-X-Thr/Ser) is present. The predicted pI value of 4.5 identifies FLXPER2 as an anionic peroxidase. Northern blot analysis shows that its mRNA expression is unique to stem tissue. Amino acid sequence comparisons show high similarity between FLXPER2 and peanut, rice, and tobacco peroxidases.

Four peroxidase (EC 1.11.1.7) isozymes were isolated from each of two flax genotrophs. All four isozymes were glycoproteins and all exhibited indoleacetic acid (IAA) oxidase activity. The percentage purity of two of the isozymes was very high; these isozymes differed in percentage carbohydrate and in peroxidase and IAA oxidase specific activities. Three of the isozymes displayed molecular weight values of about 43 000; for the fourth, molecular weight was considerably higher. Corresponding isozymes from the genotrophs and from two other flax genotypes displayed molecular weight differences which corresponded to electrophoretic relative mobility differences. Enzyme yield per unit fresh weight was higher for one genotroph than the other, and the balance between peroxidase activity and IAA oxidase activity between the genotrophs was different.

ABSTRACIIn the flax (Linum usitatissimum) genotype Stormont cirrus, anodic peroxidases from the genotroph S migrate more slowly on PAGE and SDS-PAGE than the corresponding peroxidases from the genotroph L. When purified isoperoxidases S2 and L2 were digested with a-mannosidase, the difference in mobility was eliminated. Treatment with a-fucosidase and j8-xylosidase also altered the mobility of S2 and L2, but affected the sensitivity to the action of endo-O-N-acetylglucosaminidase H of only S2. Our results suggest differences in posttranslational processing of the carbohydrate moiety between S and L isoperoxidases. These differences were also found in other S and L glycoenzymes (anodic acid phosphatases) as well as in the peroxidases of other flax genotypes.Durrant (8,9) Rm value of the L type (11, 13). These observations suggest that the Rm shifts are the result of posttranslational modifications since it is unlikely that changes across all possible structural gene loci for these enzymes would yield unidirectional Rm shifts. The absence ofco-dominance in F, hybrids ofL and S for all isozymes of peroxidase (24) also points to differences in posttranslational processing. An obvious common site for posttranslational modification of both the peroxidase and acid phosphatase isozymes in genotrophs L and S is their carbohydrate moiety since they all are glycoproteins (14). This is further supported by the recent observations that corresponding L and S isoperoxidases can be chromatographically separated from one another on columns of Sepharose-bound Con A (15) but not by isoelectric focusing nor can they be distinguished from one another by immunochemical methods (16).In this report, we present results from investigations into the structure of the carbohydrate moiety of purified L and S peroxidase isozymes using column chromatography on several Sepharose-bound lectins with different sugar affinities and the effect of various glycosidase digestions on the Rm difference between L and S peroxidase and acid phosphatase isozymes. The anodic peroxidase pattern in several other flax genotypes was also examined along with the effect of a-mannosidase treatment on their migration on PAGE. Our results suggest that the Rm differences between L and S glycoenzymes are due to higher levels of mannosylation of these proteins in the genotroph S. MATERIALS AND METHODS

Relative mobilities (Rm's) of peroxidase and acid phosphatase isozymes were examined in leaves of flax (Linum usitatissimum L.). The leaves were sampled from four equidistantly spaced positions from main stem base to apex in various genotypes. Rm's for the two slowest-migrating isozymes of each enzyme system changed in a simple, coherent fashion in leaves from stem bases toward apices. The Rm trends up the stem seen in two highly branched flax types were somewhat different from those in two sparsely branched types. The coherent Rm trends in the four types, suggesting a smooth continuum and a potentially large number of slightly different forms of these isozymes, are discussed in relation to other data for such Rm trends. In the study reported here, both enzyme systems behaved similarly. This fact and the simple Mendelian genetical system with no codominance controlling Rm's in flax peroxidases and acid phosphatases suggest posttranscriptional or posttranslational modifications as plausible mechanisms underlying the numerous, presumably small molecular changes generating the small, consistent changes in Rm's.