Antifreeze protein produced endogenously in winter rye leaves

After cold acclimation, winter rye (Secale cereale L.) is able to withstand the formation of extracellular ice at freezing temperatures. We now show, for the first time, that cold-acclimated winter rye plants contain endogenously produced antifreeze protein. The protein was extracted from the apoplast of winter rye leaves, where ice forms during freezing. After partial purification, the protein was identified as antifreeze protein because it modified the normal growth pattern of ice crystals and depressed the freezing temperature of water noncolligatively.     

Biosynthesis of winter flounder antifreeze proprotein in E.coli

A semisynthetic winter flounder antifreeze proprotein (proAFP) coding region was constructed and inserted into a lacZ expression vector. ProAFP was produced from the vector in Escherichia coli as a C-terminal fusion to the first 289 amino acids of beta-galactosidase (beta-gal). The proAFP and beta-gal domains of the beta-gal-proAFP fusion protein were separated by the recognition signal for the blood coagulation protease, factor Xa. Upon induction with isopropylthio-beta-D-galactoside the fusion protein accumulated to levels of 15% of the total protein. The beta-gal-proAFP fusion protein was partially purified by differential centrifugation, but required solubilization prior to factor Xa digestion. The solubilized fusion protein was efficiently and correctly cleaved by factor Xa, after which the proAFP was purified by gel permeation. Bacterial proAFP was indistinguishable from natural proAFP by the criteria of antifreeze activity, amino-terminal sequence (15 cycles), reverse-phase HPLC and SDS-polyacrylamide gel electrophoresis. Circular dichroism measurements showed that proAFP is a composite of random coil and alpha-helical secondary structure, with an alpha-helix content of 44% at 0 degrees C. It seems probable that the C-terminal region of proAFP, which corresponds to the mature AFP protein, is mainly alpha-helical, and that the N-terminal pro-segment is random coiled.     

Vernalization and epigenetics: how plants remember winter

One of the remarkable aspects of the promotion of flowering by vernalization is that plants have evolved the ability to measure a complete winter season of cold and to 'remember' this prior cold exposure in the spring. Recent work in Arabidopsis demonstrates the molecular basis of this memory of winter: vernalization causes changes in the chromatin structure of a flowering repressor gene, FLOWERING LOCUS C (FLC), that switch this gene into a repressed state that is mitotically stable. A key component of the vernalization pathway, VERNALIZATION INSENSITIVE3 (VIN3), which is a PHD-domain-containing protein, is induced only after a prolonged period of cold. VIN3 is involved in initiating the modification of FLC chromatin structure. The stable silencing of FLC also requires the DNA-binding protein VERNALIZATION1 (VRN1) and the polycomb-group protein VRN2.     

Antifreeze proteins in winter rye leaves form oligomeric complexes

Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides.     

Cold-induced accumulation of protein in the leaves of spring and winter barley cultivars

Electrophoretic pattern and quantitative changes in soluble proteins were determined in the leaves of spring and winter cultivars of barley (Hordeum vulgare L., cv. Makouei and cv. Reyhan, respectively) exposed to 4 degrees C for 14 d. Seedlings were grown in a controlled growth chamber for 2 weeks at a constant air temperature of 20 degrees C and then transferred to constant 4 degrees C for 14 d followed by returning to 20 degrees C (cold treatment), or they were maintained throughout at 20 degrees C during the experimental period of 40 d (control treatment). Plants were sampled every 48 h for leaf fresh weight measurements. Total leaf soluble proteins were extracted and their concentration was either determined by a colorimetric method, or size-fractionated on SDS-PAGE. Low temperature-induced increases in protein amount occurred over the second week of exposure to cold treatment irrespective of cultivar: the winter cultivar was 2 d prior in this response. The protein patterns and their density showed differences between-cultivars and between-temperature treatments. A new cold-induced polypeptide was recognized in the leaves of winter barley cultivar on day 22 (8 d at 4 degrees C) compared to the control. This polypeptide was produced earlier over the first 48 h of low temperature in the winter cultivar compared with the spring one, recognizing in the leaves of cold-treated seedling until day 26. This more rapid response to a low temperature by the winter barley cultivar indicates a more sensitive response compared with the spring barley, probably cold-shock protein is a component of this cold-induced response.     

The comparison of proteins with immunochemical affinity to stress protein 310 kD in cytoplasmatic proteins of winter rye,winter wheat,Elymus and maize

The search for proteins, immunochemically related to winter rye CSP 310 among the native cytoplasmatic proteins of a number of cultivated cereals with different tolerance to low temperatures—maize, winter wheat and winter rye and the very low temperature tolerant wild grass—Elymus sibiricus was carried out. Western blotting showed that among the native cytoplasmatic proteins of all species investigated there are proteins immunochemically related to CSP 310 protein with molecular weights about 230 and about 140–110 kD. Proteins with molecular weights about 480 and 310 kD were found in significant amounts only in winter rye. In E. sibiricus proteins with molecular weights 380–320 kD were present but these were not present among the cytoplasmatic protein spectra of the other species. In each case the proteins immunochemically related to CSP 310 consisted of different combinations of two types of subunits.     

The effect of temperature elevation and feeding on the pancreas of Rana esculenta in late winter

Summary Hibernating frogs (Rana esculenta) in late winter were subjected to a higher environmental temperature. In addition the animals were fed. Electronmicroscopical and biochemical methods were used to follow the changes in the pancreas under these circumstances.The frog pancreas in winter is characterized by a very low protein synthesizing capacity, while the Golgi system is an extremely compact structure, composed of a mass of small vesicles.A rise in the environmental temperature does not initiate protein synthesis. However, zymogen granules appear and the Golgi system becomes more differentiated.Under these conditions protein synthesis is not started until a few hours after feeding. This is not accompanied by striking structural changes.     

The synthesis of freezing-point-depressing protein of the winter flounder Pseudopleuronectusamericanus in Xenpuslaevis oocytes

The serum of the winter flounder $\text{Pseudopleuronectus}$$\text{americanus}$ contains a freezing-point-depressing protein of a molecular weight approximately 10,000 with 60% alanine in its composition. When injected into Xenopus o?cyte, a 6–10 S, poly A-rich RNA preparation isolated from the fish liver polysomes stimulated 3–4 fold the incorporation of [³H] alanine into 10% trichloroacetic acid-soluble, non-dialysable proteins. Analysis of the protein fractions showed a translation product similar in molecular weight and electrophoretic mobility to flounder freezing-point-depressing protein. These observations indicated that the 6–10 S RNA from the flounder contained mRNA for the synthesis of flounder's freezing-point-depressing protein.     

小麦品种蛋白质品质性状稳定性研究

用陕西省关中小麦品种区域试验所选用的12个小麦品种（品系）在12个试点的数据资料。分析了品种,环境及品种与环境互作（CEI）对籽粒硬度,蛋白质含量,沉淀值及湿面筋含量的影响。结果表明：基因型效应对所有品质参数均有显著影响,基因型与环境互作对沉淀值影响较大,而对籽粒硬度,蛋白质含量与湿面筋含量影响较小,环境效应对湿面筋含量和好粒硬度影响较大,而对蛋白质含量与沉淀值影响较小,蛋白质品质参数的回归系数（b值）表明,基因型对不同环境的反应存在着显著差异。对于籽粒硬度,蛋白质含量与湿面筋含量回归偏差显著的品种很少,这表明线性回归模式占了基因型变异的绝大部分,一些品种的沉淀值显著偏离了回归,上述结果表明,要改善小麦品种的蛋白质品质,也应重视环境对小麦蛋白质品质的影响。     

干旱和ABA对同核异质冬小麦叶片蛋白的影响

利用双向凝胶电泳研究了冬小麦核质杂种NC4、NC37和它们的核供体丰抗13的3个品种幼苗在水分胁迫和外施ABA条件下叶片中蛋白质代谢的变化。结果表明水分胁迫可抑制3种小麦叶片中一些蛋白质合成,使蛋白数量减少,而在NC4、NC37两个核质杂种中有1个PI5.8、20kD的新合成蛋白点出现,根部外伤ABA也可诱导该蛋白合成,核供体丰抗13幼苗中,ABA可诱导合成该蛋白,而水分胁迫时该蛋白没有出现,表明该蛋白由核基因编码,而其表达可能由细胞质中与ABA有关的某种机制调控。     

Abscisic acid-induced secretory proteins in suspension-cultured cells of winter wheat

In suspension-cultured cells of winter wheat (Triticum aestivum L. cv. Chihokukomugi), the accumulation of soluble secretory proteins in the culture medium was promoted by ABA treatment in comparison with non-treated cells. The total amount of secretory proteins in ABA-treated cells was 1.7-fold higher than that in non-treated cells. The analysis of two-dimensional electrophoresis revealed that at least twelve secretory proteins were induced by ABA, and these were named WAS (wheat ABA-induced secretory) proteins 1 to 12. N-terminal amino acid sequence analysis of WAS proteins revealed the sequences of WAS-2 and WAS-3. Homology searches showed that WAS-2 had 55% identity with the N-terminus of the wheat chemically induced gene (WCI-5 gene) product. WAS-3 was also shown to have 93% identity with the N-terminus of the barley protein R, a typical member of thaumatin-like proteins (TLPs). Immunoblot analysis also suggested that WAS-3 was related to protein R. These results suggest that exogenous ABA induces some basic secretory proteins that are related to the plant defense system in wheat.     

Liver-specific and seasonal expression of transgenic Atlantic salmon harboring the winter flounder antifreeze protein gene

We have analyzed the inheritance and expression of a line of transgenic salmon harboring the antifreeze protein gene from the winter flounder. The genomic clone 2A-7 coding for a major liver-type antifreeze protein gene (wflAFP-6) was integrated into the salmon genome. From a transgenic founder (# 1469), an F3 generation was produced. In this study, southern blot analysis showed that only one copy of the antifreeze protein transgene was integrated into a unique site in F3 transgenic fish. The integration site was cloned and characterized. Northern analysis indicated that the antifreeze protein mRNA was only expressed in the liver and showed seasonal variation. All of the F3 offspring contained similar levels of the antifreeze protein precursor protein in the sera and the sera of these offspring showed a characteristic hexagonal ice crystal pattern indicating the presence of antifreeze activity. In addition, the antifreeze protein precursor protein level was found to vary with the season, being highest in the month of November and lowest in May. This study had demonstrated a tissue-specific and stable expression of the antifreeze protein transgene in the F3 generation of the transgenic salmon 1469 line.     

In vitro protein synthesis during germination and vernalization in winter wheat embryos

Protein synthesis during germination at 24?C and vernalizationat 4?C in winter wheat embryos were investigated with a cell-freesystem. During germination, the capacity for protein synthesisincreased in the early stage between 12 and 36 hr of imbibitionthen declined to a final low level between 48 and 72 hr. Thistransition was due to quantitative changes of the activitiesof ribosomal and supernatant fractions in the early stage andmainly to those of the supernatant fraction in the later stage.During vernalization, the capacity for protein synthesis continuedto decline over 15 to 60 days at 4?C. This transition was dueto the change in activity of the supernatant fraction; the activityof the ribosomal fraction was nearly constant. Electrophoretic analysis of in vitro products indicated thatthe high molecular weight proteins present in 12-hr embryoshad disappeared in 48-hr germinated wheat embryos and that theproducts in 24- and 36-hr embryos were types intermediate betweenthose of 12- and 48-hr embryos. The products in each vernalizedembryo resembled those in 24- and 36-hr germinated embryos.Therefore, it was concluded that the mRNA species for translationchanged during germination and vernalization in winter wheatembryos. (Received January 20, 1977; )     

The effect of hypothermia on the content of 310 kD stress protein in seedlings of winter rye and wheat

The effect of hypothermia on the content of 310 kD stress protein in seedlings of winter rye and wheat was studied by rocket-immunoelectrophoresis and radioactive label. The 1-h low-temperature stress was found to result in an increase in the content of this protein at both above- and below-zero temperatures. It was found that the increase in the relative content of the protein with mol. wt 310 kD, under the effect of short-term low-temperature stress, occurs due to induction of its synthesis. It has been found that during cold hardening of winter wheat the content of this protein decreases up to 64% compared to the control during the first day of hardening but starts to rise by the third day and reaches 179% by the seventh day, which is well correlated with the increase in cold resistance of winter wheat plants during cold hardening.     

Chemical composition of antarctic zooplankton during austral fall and winter

Water level, ash content, proximate (protein, lipid, carbohydrate and chitin) and elemental (carbon and nitrogen) composition were analyzed in twentythree species of Antarctic Zooplankton collected during the austral fall (1986) and winter (1988) from the Scotia/Weddell Sea region. Extremes in water level, ash content and organic components were typified by copepods and gelatinous forms. Ostracods and polychaetes were generally similar in composition to copepods, being only slightly higher in water level and ash content. Chaetognaths exhibited a composition intermediate in character with some components similar in value to that shown by crustaceans (i.e. protein) while other components were more in the range of values seen in gelatinous forms (i.e. water level and ash content). Protein was the major proximate component and measured values (as % Afdw) were fairly uniform among non-gelatinous species (x=33.9±6.9). Lipid levels were variable, with high values (>30% AFDW) only found for the copepods Calanoides acutus, Calanus propinquus and Euchaeta antarctica. Carbohydrate values were low in all species examined. Chitin was measured in crustacean species only. With the exception of C. acutus (x=2.5% AFDW chitin), values were similar among species with mean values being slightly higher in fall (x=11.8±2.5) than in winter (x=6.7±1.8). Among non-gelatinous species, the ratio of carbon to nitrogen was positively correlated with the lipid to protein ratio, underscoring the compositional association between elemental and proximate components in these groups. In gelatinous species, the relationship between carbon:nitrogen and lipid:protein was inconsistent and less pronounced. Caloric content was estimated from recovered organic matter for nongelatinous species. As a function of wet weight and dry weight, values reflected differences in water level and ash content among individual species. As a function of ashfree dry weight, values were similar among all species (x=3.6±0.9 kcal/g).Seasonal comparisons were possible for 12 of the 23 species. Among crustaceans, changes in water level and organic components were variable reflecting dissimilar trophic, reproductive or ecological habits among different species. Essentially no change in composition between fall and winter was observed for diapause species (e.g. Calanoides acutus and Rhincalanus gigas) as well as for omnivorous/ carnivorous species (e.g. Gaetanus tenuispinus). Conversely, large compositional changes were evident for Calanus propinquus, a small-particle grazer that relies heavily on lipid reserves. Chaetognaths and some gelatinous species exhibited a considerable decrease in ash content from fall to winter which, for most cases, was mirrored by some degree of increase in lipid level. At present, however, scant data are available to help explain the observed patterns of compositional change within non-crustacean species.     

Cellular localization of pleurocidin gene expression and synthesis in winter flounder gill using immunohistochemistry and in situ hybridization

Pleurocidin is an antimicrobial peptide isolated from winter flounder and has been previously localized to mucous cells of the skin epidermis and the intestine. The present study was designed to determine the cell type involved in pleurocidin gene expression and protein synthesis in gills from the same species. Whole-mount in situ hybridization with a pleurocidin-specific RNA probe and whole-mount immunohistochemistry with an anti-pleurocidin antibody localized the expression of this gene and the synthesis of its corresponding protein in a population of cells primarily isolated to the non-lamellar portion of the gill filament. Histological techniques allowed the presumptive identification of these cells as eosinophilic granular cells. These observations suggest that pleurocidin is expressed in not one but two distinct populations of cells within the winter flounder, one being an important group of inflammatory cells, the eosinophilic granular cells.     

Coregulation of light-harvesting complex II phosphorylation and lhcb mRNA accumulation in winter rye

Winter rye plants grown under contrasting environmental conditions or just transiently shifted to varying light and temperature conditions, were studied to elucidate the chloroplast signal involved in regulation of photosynthesis genes in the nucleus. Photosystem II excitation pressure, reflecting the plastoquinone redox state, and the phosphorylation level of thylakoid light-harvesting proteins (LHCII and CP29) were monitored together with changes occurring in the accumulation of lhcb, rbcS and psbA mRNAs. Short-term shifts of plants to changed conditions, from 1 h to 2 d, were postulated to reveal signals crucial for the initiation of the acclimation process. Comparison of these results with those obtained from plants acclimated during several weeks' growth at contrasting temperature and in different light regimes, allow us to make the following conclusions: (1) LHCII protein phosphoylation is a sensitive tool to monitor redox changes in chloroplasts; (2) LHCII protein phosphorylation and lhcb mRNA accumulation occur under similar conditions and are possibly coregulated via an activation state of the same kinase (the LHCII kinase); (3) Maximal accumulation of lhcb mRNA during the diurnal light phase seems to require an active LHCII kinase whereas inactivation of the kinase is accompanied by dampening of the circadian oscillation in the amount of lhcb mRNA; (4) Excitation pressure of photosystem II (reduction state of the plastoquinone pool) is not directly involved in the regulation of lhcb mRNA accumulation. Instead (5) the redox status of the electron acceptors of photosystem I in the stromal compartment seems to be highly regulated and crucial for the regulation of lhcb gene expression in the nucleus.