Purification, characterization, and structural analysis of a plant low-temperature-induced protein.

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 low-temperature-induced proteins, BN28 is extremely hydrophilic, such that it remains soluble following boiling. Immunoblot 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 (100% coil) in aqueous solution. Although it had earlier been speculated that BN28-like proteins from Arabidopsis thaliana might possess antifreeze protein activity (S. Kurkela and M. Franck [1990] Plant Mol Biol 15: 137-144), no such activity could be detected in ice recrystallization assays with rBN28.

Inheritance and expression patterns of BN28, a low temperature induced gene in Brassica napus, throughout the Brassicaceae.

Molecular genetics is becoming an important tool in the breeding and selection of agronomically important traits. BN28 is a low temperature induced gene in Brassicaceae species. PCR and Southern blot analysis indicate that BN28 is polymorphic in the three diploid genomes: Brassica rapa (AA), Brassica nigra (BB), and Brassica oleracea (CC). Of the allotetraploids, Brassica napus (AACC) is the only species to have inherited homologous genes from both parental genomes. Brassica juncea (AABB) and Brassica carinata (BBCC) have inherited homologues from the AA and CC genomes, respectively, while Sinapsis arvensis (SS) contains a single homologue from the BB genome and Sinapsis alba (dd) appears to be different from all the diploid parents. All species show message induction when exposed to low temperature. However, differences in expression were noticed at the protein level, with silencing occurring in the BB genome at the level of translation. Results suggest that silencing is occurring in diploid species where duplication may not have occurred. Molecular characterization and inheritance of BN28 homologues in the Brassicaceae may play an important role in determining their quantitative function during exposure to low temperature. Key words : Brassicaceae, BN28, inheritance, polymorphism.

Expression of a Low-Temperature-Induced Protein in Brassica napus.

BN28 is a low-temperature-induced, boiling-soluble protein in Brassica napus. We used antibodies raised against a recombinant BN28 to examine the expression of this protein in cold-acclimating plants and to investigate its relationship to plant freezing tolerance. Changes in the steady-state levels of BN28 protein appear to lag several days behind those of the mRNA. BN28 is first detected on immunoblots after approximately 8 d of exposure to low temperature, and thereafter levels remain stable while plants are maintained at 4[deg]C. Radiolabeling studies indicate that BN28 is synthesized at a relatively low rate. A decline in protein levels is observed soon after returning plants to control temperatures, and little or no protein can be detected after 7 d of deacclimation. The disappearance of the protein precedes a loss in freezing tolerance, suggesting that BN28 is not involved in maintaining plasma membrane integrity. Expression of BN28 is observed primarily in leaves and appears to be low-temperature specific. Quantitative analysis indicated that BN28 accumulates to approximately 82.7 pmol mg-1 total protein in cold-acclimated leaves. This concentration is similar to that reported for two group 2 late-embryogenesis-abundant-like proteins.

Ethylene Production during Development of Mustard (Brassica juncea) and Canola (Brassica napus) Seed.

An open, continuous flow system was used to investigate ethylene production during degreening of maturing seed of mustard (Brassica juncea cv Cutlass and cv Lethbridge 22A) and canola (Brassica napus cv Westar and cv Alto). Isolated mustard seed evolved higher amounts of ethylene than those of canola, and this was particularly evident both early in embryogeny and later during the desiccation phase of seed maturation. The silique walls produced negligible amounts of ethylene in both species. The concentrations of ethylene surrounding seed as they matured within siliques were significantly higher in mustard than in canola, and this interspecies difference was greatest during the seed desiccation phase. In mustard, a 4-fold increase in silique internal ethylene levels was apparent during desiccation. In comparison, only a moderate increase in silique-derived ethylene occurred in canola.

Frost, Abscisic Acid, and Desiccation Hasten Embryo Development in Brassica napus.

Seed development in canola (Brassica napus) following a mild nonlethal freeze was examined with respect to abscisic acid (ABA) levels, desiccation, and expression of LEA.76 and isocitrate lyase (ICL) transcripts. Plants with seed of 70 and 55% moisture contents were frozen to -5 degrees C for 3 hours, and seed development followed after thawing. In addition, similar processes were compared during induction of extreme desiccation tolerance by application of ABA in Brassica microspore-derived haploid embryos in culture. A mild freeze/thaw caused a premature switch in seed developmental direction from predesiccation to desiccation as indicated by an immediate and accelerated loss of seed moisture to levels similar to the mature seed in 7 instead of 35 days, and by elevated ABA levels and induction of low levels of LEA.76 and ICL transcripts. Similarly, addition of ABA to haploid embryos in culture resulted in the induction of desiccation tolerance and low levels of late embryogenesis-abundant (LEA) but not ICL transcripts. In contrast, normal seed development and desiccation of ABA-treated (desiccation-tolerant) embryos resulted in the induction of ICL and very high levels of LEA.76 transcripts. Similarly, desiccation of control (desiccation-sensitive) embryos resulted in very high levels of LEA.76 transcripts. These results indicate that although LEA-type proteins have been implicated in the development of desiccation tolerance, high transcript levels of LEA.76 were not observed in the induction of desiccation tolerance either by a hastening of the maturation process in the developing Brassica seed, or by the exogenous application of ABA to Brassica haploid embryos in culture.

Role of Abscisic Acid in the Induction of Freezing Tolerance in Brassica napus Suspension-Cultured Cells.

Brassica napus suspension-cultured cells could be hardened in 6 days at 25 degrees C by the addition of mefluidide or ABA to the culture medium. Cells treated with mefluidide (10 milligrams per liter) or ABA (50 micromolar) attained an LT(50) of -17.5 degrees C or -18 degrees C, respectively, while the LT(50) for the comparable nonhardened control (sucrose) was -10 degrees C. The increased freezing tolerance of mefluidide-treated cells was paralleled by a 4- to 23-fold increase in ABA, as measured by gas-liquid chromatography using electron capture detection. Application of 1 milligram per liter of fluridone, an inhibitor of abscisic acid biosynthesis, prevented the mefluidide-induced increase in freezing tolerance and the accumulation of ABA. Both these inhibitory effects of fluridone were overridden by 50 micromolar ABA in the culture medium. On the basis of these results, we concluded that increased ABA levels are important for the induction of freezing tolerance in suspension-cultured cells.

Induction of freezing tolerance in microspore-derived embryos of winter Brassica napus.

Freezing tolerance was induced in microspore derived embryos of winter Brassica napus cv. Jet neuf by the addition of ABA or mefluidide to the culture media during embryogenesis. Survival after freezing was estimated by culture of frozen-thawed embryos to plantlets. A higher freezing tolerance (50% survival at -15°C) was induced when 50 µM ABA or 3.2 µM mefluidide was incorporated initially into the medium during embryogenesis at 25°C followed by culture at 2°C for 3 weeks. When embryos were induced in the absence of ABA or mefluidide and maintained at 2°C for even as long as 12 weeks a lower degree of freezing tolerance (10% survival at -15°C) was obtained. Plants regenerated from embryos hardened maximally by a combination of either ABA or MFD with low temperature did not require further vernalization for flowering.

Alteration of Gene Expression during the Induction of Freezing Tolerance in Brassica napus Suspension Cultures.

Brassica napus suspension-cultured cells can be hardened to a lethal temperature for 50% of the sample of -20 degrees C in eight days at room temperature with abscisic acid. During the induction of freezing tolerance, changes were observed in the electrophoretic pattern of [(35)S]methionine labeled polypeptides. In hardening cells, a 20 kilodalton polypeptide was induced on day 2 and its level increased during hardening. The induction of freezing tolerance with nonmaximal hardening regimens also resulted in increases in the 20 kilodalton polypeptide. The 20 kilodalton polypeptide was associated with a membrane fraction enriched in endoplasmic reticulum and was resolved as a single spot by two-dimensional electrophoresis. In vitro translation of mRNA indicate alteration of gene expression during abscisic acid induction of freezing tolerance. The new mRNA encodes a 20 kilodalton polypeptide associated with increased freezing tolerance induced by either abscisic acid or high sucrose. A 20 kilodalton polypeptide was also translated by mRNA isolated from cold-hardened B. napus plants.

Root Respiration in White Spruce (Picea glauca [Moench] Voss) Seedlings in Relation to Morphology and Environment.

Roots of white spruce seedlings (Picea glauca [Moench] Voss) undergo respiratory changes during the year that are related to changing metabolic requirements. An alternative pathway is always present, functions during most of the year, and operates maximally during periods of root and shoot growth. Although some differences in respiration and the apportioning of respiration can be correlated to root morphology, the environment and the stage of shoot development are also important controls. Differences in respiration related to root morphology are not manifest in mitochondrial structure, but overall rates were found to correlate with the number of mitochondria present. Root respiration in seedlings grown under root growth capacity conditions reflects root and shoot growth at that time rather than root growth capacity.

Peroxidase Activity in Relation to Suberization and Respiration in White Spruce (Picea glauca [Moench] Voss) Seedling Roots.

Peroxidase (EC 1.11.1.7) activity is associated with suberization during endodermal development and metacutization in roots of white spruce (Picea glauca [Moench] Voss) seedlings. Histochemical analysis indicates a relationship between suberization and peroxidase activity, but peroxidase is ubiquitous. Increased peroxidase activity results from the induction of four anodic peroxidase isozymes in addition to quantitative increases in two anodic peroxidase isozymes. Four of these polymerized eugenol. Cold temperatures induce formation of two anodic isozymes and result in suberization. The increased peroxidase activity associated with suberization is correlated to residual respiration. In an attempt to elucidate this relationship, the effect of respiratory inhibitors on respiration and peroxidase activity are compared.

The effect of rotenone on respiration in pea cotyledon mitochondria.

Respiration utilizing NAD-linked substrates in mitochondria isolated from cotyledons of etiolated peas (Pisum sativum L. var. Homesteader) by sucrose density gradient centrifugation exhibited resistance to rotenone. The inhibited rate of alpha-ketoglutarate oxidation was equivalent to the recovered rate of malate oxidation. (The recovered rate is the rate following the transient inhibition by rotenone.) The inhibitory effect of rotenone on malate oxidation increased with increasing respiratory control ratios as the mitochondria developed. The cyanide-resistant and rotenone-resistant pathways followed different courses of development as cotyledons aged. The rotenone-resistant pathway transferred reducing equivalents to the cyanide-sensitive pathway. Malic enzyme was found to be inhibited competitively with respect to NAD by rotenone concentrations as low as 1.67 micromolar. In pea cotyledon mitochondria, rotenone was transformed into elliptone. This reduced its inhibitory effect on intact mitochondria. Malate dehydrogenase was not affected by rotenone or elliptone. However, elliptone inhibited malic enzyme to the same extent that rotenone did when NAD was the cofactor. The products of malate oxidation reflected the interaction between malic enzyme and malate dehydrogenase. Rotenone also inhibited the NADH dehydrogenase associated with malate dehydrogenase. Thus, rotenone seemed to exert its inhibitory effect on two enzymes of the electron transport chain of pea cotyledon mitochondria.