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Celiac disease is the most common food-induced enteropathy in humans, with a prevalence of approximately 1% worldwide. It is induced by digestion-resistant, proline- and glutamine-rich seed storage proteins, collectively referred to as gluten, found in wheat (Triticum aestivum). Related prolamins are present in barley (Hordeum vulgare) and rye (Secale cereale). The incidence of both celiac disease and a related condition called nonceliac gluten sensitivity is increasing. This has prompted efforts to identify methods of lowering gluten in wheat, one of the most important cereal crops. Here, we used bulked segregant RNA sequencing and map-based cloning to identify the genetic lesion underlying a recessive, low-prolamin mutation (lys3a) in diploid barley. We confirmed the mutant identity by complementing the lys3a mutant with a transgenic copy of the wild-type barley gene and then used targeting-induced local lesions in genomes to identify induced single-nucleotide polymorphisms in the three homeologs of the corresponding wheat gene. Combining inactivating mutations in the three subgenomes of hexaploid bread wheat in a single wheat line lowered gliadin and low-molecular-weight glutenin accumulation by 50% to 60% and increased free and protein-bound lysine by 33%.

Benzoxazinones (Bxs) are major defensive secondary metabolites in wheat (Triticum aestivum), rye (Secale cereale), and maize (Zea mays). Here, we identified full sets of homeologous and paralogous genes encoding Bx glucosyltransferase (GT) and Bx-glucoside glucosidase (Glu) in hexaploid wheat (2n = 6x = 42; AABBDD). Four GT loci (TaGTa–TaGTd) were mapped on chromosomes 7A, 7B (two loci), and 7D, whereas four glu1 loci (Taglu1a–Taglu1d) were on chromosomes 2A, 2B (two loci), and 2D. Transcript levels differed greatly among the four loci; B-genome loci of both TaGT and Taglu1 genes were preferentially transcribed. Catalytic properties of the enzyme encoded by each homeolog/paralog also differed despite high levels of identity among amino acid sequences. The predominant contribution of the B genome to GT and Glu reactions was revealed, as observed previously for the five Bx biosynthetic genes, TaBx1 to TaBx5, which are separately located on homeologous groups 4 and 5 chromosomes. In rye, where the ScBx1 to ScBx5 genes are dispersed to chromosomes 7R and 5R, ScGT and Scglu were located separately on chromosomes 4R and 2R, respectively. The dispersal of Bx-pathway loci to four distinct chromosomes in hexaploid wheat and rye suggests that the clustering of Bx-pathway genes, as found in maize, is not essential for coordinated transcription. On the other hand, barley (Hordeum vulgare) was found to lack the orthologous GT and glu loci like the Bx1 to Bx5 loci despite its close phylogenetic relationship with wheat and rye. These results contribute to our understanding of the evolutionary processes that the Bx-pathway loci have undergone in grasses.

The effector protein NIP1 from the barley (Hordeum vulgare) pathogen Rhynchosporium secalis specifically induces the synthesis of defense-related proteins in cultivars of barley expressing the complementary resistance gene, Rrs1. In addition, it stimulates the activity of the barley plasma membrane H(+)-ATPase in a genotype-unspecific manner and it induces necrotic lesions in leaf tissues of barley and other cereal plant species. NIP1 variants type I and II, which display quantitative differences in their activities as elicitor and H(+)-ATPase stimulator, and the inactive mutant variants type III* and type IV*, were produced in Escherichia coli. Binding studies using (125)I-NIP1 type I revealed a single class of binding sites with identical binding characteristics in microsomes from near-isogenic resistant (Rrs1) and susceptible (rrs1) barley. Binding was specific, reversible, and saturable, and saturation ligand-binding experiments yielded a K(d) of 5.6 nm. A binding site was also found in rye (Secale cereale) and the nonhost species wheat (Triticum aestivum), oat (Avena sativa), and maize (Zea mays), but not in Arabidopsis (Arabidopsis thaliana). For NIP1 types I and II, equilibrium competition-binding experiments revealed a correlation between the difference in their affinities to the binding site and the differences in their elicitor activity and H(+)-ATPase stimulation, indicating a single target molecule to mediate both activities. In contrast, the inactive proteins type III* and type IV* are both characterized by high affinities similar to type I, suggesting that binding of NIP1 to this target is not sufficient for its activities.

Extracellular pathogenesis-related proteins, including glucanases, are expressed at cold temperatures in winter rye (Secale cereale) and display antifreeze activity. We have characterized recombinant cold-induced glucanases from winter rye to further examine their roles and contributions to cold tolerance. Both basic β-1,3-glucanases and an acidic β-1,3;1,4-glucanase were expressed in Escherichia coli, purified, and assayed for their hydrolytic and antifreeze activities in vitro. All were found to be cold active and to retain partial hydrolytic activity at subzero temperatures (e.g. 14%–35% at −4°C). The two types of glucanases had antifreeze activity as measured by their ability to modify the growth of ice crystals. Structural models for the winter rye β-1,3-glucanases were developed on which putative ice-binding surfaces (IBSs) were identified. Residues on the putative IBSs were charge conserved for each of the expressed glucanases, with the exception of one β-1,3-glucanase recovered from nonacclimated winter rye in which a charged amino acid was present on the putative IBS. This protein also had a reduced antifreeze activity relative to the other expressed glucanases. These results support the hypothesis that winter rye glucanases have evolved to inhibit the formation of large, potentially fatal ice crystals, in addition to having enzymatic activity with a potential role in resisting infection by psychrophilic pathogens. Glucanases of winter rye provide an interesting example of protein evolution and adaptation aimed to combat cold and freezing conditions.

The wheat (Triticum aestivum) and rye (Secale cereale) β-d-glucosidases hydrolyze hydroxamic acid-glucose conjugates, exist as different types of isozyme, and function as oligomers. In this study, three cDNAs encoding β-d-glucosidases (TaGlu1a, TaGlu1b, and TaGlu1c) were isolated from young wheat shoots. Although the TaGlu1s share very high sequence homology, the mRNA level of Taglu1c was much lower than the other two genes in 48- and 96-h-old wheat shoots. The expression ratio of each gene was different between two wheat cultivars. Recombinant TaGlu1b expressed in Escherichia coli was electrophoretically distinct fromTaGlu1a and TaGlu1c. Furthermore, coexpression of TaGlu1a and TaGlu1b gave seven bands on a native-PAGE gel, indicating the formation of both homo- and heterohexamers. One distinctive property of the wheat and rye glucosidases is that they function as hexamers but lose activity when dissociated into smaller oligomers or monomers. The crystal structure of hexameric TaGlu1b was determined at a resolution of 1.8 Å. The N-terminal region was located at the dimer-dimer interface and plays a crucial role in hexamer formation. Mutational analyses revealed that the aromatic side chain at position 378, which is located at the entrance to the catalytic center, plays an important role in substrate binding. Additionally, serine-464 and leucine-465 of TaGlu1a were shown to be critical in the relative specificity for DIMBOA-glucose (2-O-β-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) over DIBOA-glucose (7-demethoxy-DIMBOA-glucose).

GrainGenes (http://wheat.pw.usda.gov) is an international database for genetic and genomic information about Triticeae species (wheat [Triticum aestivum], barley [Hordeum vulgare], rye [Secale cereale], and their wild relatives) and oat (Avena sativa) and its wild relatives. A major strength of the GrainGenes project is the interaction of the curators with database users in the research community, placing GrainGenes as both a data repository and information hub. The primary intensively curated data classes are genetic and physical maps, probes used for mapping, classical genes, quantitative trait loci, and contact information for Triticeae and oat scientists. Curation of these classes involves important contributions from the GrainGenes community, both as primary data sources and reviewers of published data. Other partially automated data classes include literature references, sequences, and links to other databases. Beyond the GrainGenes database per se, the Web site incorporates other more specific databases, informational topics, and downloadable files. For example, unique BLAST datasets of sequences applicable to Triticeae research include mapped wheat expressed sequence tags, expressed sequence tag-derived simple sequence repeats, and repetitive sequences. In 2004, the GrainGenes project migrated from the AceDB database and separate Web site to an integrated relational database and Internet resource, a major step forward in database delivery. The process of this migration and its impacts on database curation and maintenance are described, and a perspective on how a genomic database can expedite research and crop improvement is provided.

During cold acclimation, winter rye (Secale cereale L. cv Musketeer) plants accumulate antifreeze proteins (AFPs) in the apoplast of leaves and crowns. The goal of this study was to determine whether these AFPs influence survival at subzero temperatures by modifying the freezing process or by acting as cryoprotectants. In order to inhibit the growth of ice, AFPs must be mobile so that they can bind to specific sites on the ice crystal lattice. Guttate obtained from cold-acclimated winter rye leaves exhibited antifreeze activity, indicating that the AFPs are free in solution. Infrared video thermography was used to observe freezing in winter rye leaves. In the absence of an ice nucleator, AFPs had no effect on the supercooling temperature of the leaves. However, in the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by 0.3°C to 1.2°C. In vitro studies showed that apoplastic proteins extracted from cold-acclimated winter rye leaves inhibited the recrystallization of ice and also slowed the rate of migration of ice through solution-saturated filter paper. When we examined the possible role of winter rye AFPs in cryoprotection, we found that lactate dehydrogenase activity was higher after freezing in the presence of AFPs compared with buffer, but the same effect was obtained by adding bovine serum albumin. AFPs had no effect on unstacked thylakoid volume after freezing, but did inhibit stacking of the thylakoids, thus indicating a loss of thylakoid function. We conclude that rye AFPs have no specific cryoprotective activity; rather, they interact directly with ice in planta and reduce freezing injury by slowing the growth and recrystallization of ice.

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca2+-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca2+-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca2+-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.

To understand the mechanisms responsible for aluminum (Al) toxicity and tolerance in plants, an expressed sequence tag (EST) approach was used to analyze changes in gene expression in roots of rye (Secale cereale L. cv Blanco) under Al stress. Two cDNA libraries were constructed (Al stressed and unstressed), and a total of 1,194 and 774 ESTs were generated, respectively. The putative proteins encoded by these cDNAs were uncovered by Basic Local Alignment Search Tool searches, and those ESTs showing similarity to proteins of known function were classified according to 13 different functional categories. A total of 671 known function genes were used to analyze the gene expression patterns in rye cv Blanco root tips under Al stress. Many of the previously identified Al-responsive genes showed expression differences between the libraries within 6 h of Al stress. Certain genes were selected, and their expression profiles were studied during a 48-h period using northern analysis. A total of 13 novel genes involved in cell elongation and division (tonoplast aquaporin and ubiquitin-like protein SMT3), oxidative stress (glutathione peroxidase, glucose-6-phosphate-dehydrogenase, and ascorbate peroxidase), iron metabolism (iron deficiency-specific proteins IDS3a, IDS3b, and IDS1; S-adenosyl methionine synthase; and methionine synthase), and other cellular mechanisms (pathogenesis-related protein 1.2, heme oxygenase, and epoxide hydrolase) were demonstrated to be regulated by Al stress. These genes provide new insights about the response of Al-tolerant plants to toxic levels of Al.

The light-harvesting proteins (Lhca) of photosystem I (PSI) from four monocot and five dicot species were extracted from plant material, separated by reversed-phase high-performance liquid chromatography (HPLC) and subsequently identified on the basis of their intact molecular masses upon on-line hyphenation with electrospray ionization mass spectrometry. Although their migration behavior in gel electrophoresis was very similar, the elution times among the four antenna types in reversed-phase-HPLC differed significantly, even more than those observed for the light-harvesting proteins of photosystem II. Identification of proteins is based on the good agreement between the measured intact molecular masses and the values calculated on the basis of their nucleotide-derived amino acid sequences, which makes the intact molecular masses applicable as intact mass tags. These values match excellently for Arabidopsis, most probably because of the availability of high-quality DNA sequence data. In all species examined, the four antennae eluted in the same order, namely Lhca1 > Lhca3 > Lhca4 > Lhca2. These characteristic patterns enabled an unequivocal assignment of the proteins in preparations from different species. Interestingly, in all species examined, Lhca1 and Lhca2 were present in two or three isoforms. A fifth antenna protein, corresponding to the Lhca6 gene, was found in tomato (Lycopersicon esculentum). However PSI showed a lower heterogeneity than photosystem II. In most plant species, Lhca2 and Lhca4 proteins are the most abundant PSI antenna proteins. The HPLC method used in this study was found to be highly reproducible, and the chromatograms may serve as a highly confident fingerprint for comparison within a single and among different species for future studies of the PSI antenna.

Although cold and drought adaptation in cereals and other plants involve the induction of a large number of genes, inheritance studies in Triticeae (wheat [Triticum aestivum], barley [Hordeum vulgare], and rye [Secale cereale]) have revealed only a few major loci for frost or drought tolerance that are consistent across multiple genetic backgrounds and environments. One might imagine that these loci could encode highly conserved regulatory factors that have global effects on gene expression; therefore, genes encoding central regulators identified in other plants might be orthologs of these Triticeae stress tolerance genes. The CBF/DREB1 regulators, identified originally in Arabidopsis as key components of cold and drought regulation, merit this consideration. We constructed barley cDNA libraries, screened these libraries and a barley bacterial artificial chromosome library using rice (Oryza sativa) and barley Cbf probes, found orthologs of ArabidopsisCBF/DREB1 genes, and examined the expression and genetic map location of the barley Cbf3 gene,HvCbf3. HvCbf3 was induced by a chilling treatment. HvCbf3 is located on barley chromosome 5H between markers WG364b and saflp58 on the barley cv Dicktoo × barley cv Morex genetic linkage map. This position is some 40 to 50 cM proximal to the winter hardiness quantitative trait locus that includes the Vrn-1H gene, but may coincide with the wheat 5A Rcg1 locus, which governs the threshold temperature at which cor genes are induced. From this, it remains possible that HvCbf3 is the basis of a minor quantitative trait locus in some genetic backgrounds, though that possibility remains to be thoroughly explored.

Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed.

Many plants increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Cold acclimation in Arabidopsis involves rapid cold-induced expression of the C-repeat/dehydration-responsive element binding factor (CBF) transcriptional activators followed by expression of CBF-targeted genes that increase freezing tolerance. Here, we present evidence for a CBF cold-response pathway in Brassica napus. We show thatB. napus encodes CBF-like genes and that transcripts for these genes accumulate rapidly in response to low temperature followed closely by expression of the cold-regulatedBn115 gene, an ortholog of the Arabidopsis CBF-targetedCOR15a gene. Moreover, we show that constitutive overexpression of the Arabidopsis CBF genes in transgenic B. napus plants induces expression of orthologs of Arabidopsis CBF-targeted genes and increases the freezing tolerance of both nonacclimated and cold-acclimated plants. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat (Triticum aestivumL. cv Norstar) and rye (Secale cereale L. cv Puma), which cold acclimate, as well as in tomato (Lycopersicon esculentum var. Bonny Best, Castle Mart, Micro-Tom, and D Huang), a freezing-sensitive plant that does not cold acclimate. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved amino acid sequences, PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. We conclude that components of the CBF cold-response pathway are highly conserved in flowering plants and not limited to those that cold acclimate.

Antifreeze proteins similar to two different chitinases accumulate during cold acclimation in winter rye (Secale cereale). To determine whether these cold-responsive chitinases require post-translational modification to bind to ice, cDNAs coding for two different full-length chitinases were isolated from a cDNA library produced from cold-acclimated winter rye leaves. CHT9 is a 1,193-bp clone that encodes a 31.7-kD class I chitinase andCHT46 is a 998-bp clone that codes for a 24.8-kD class II chitinase. Chitinase-antifreeze proteins purified from the plant were similar in mass to the predicted mature products ofCHT9 and CHT46, thus indicating that there was little chemical modification of the amino acid sequences in planta. To confirm these results, the mature sequences ofCHT9 and CHT46 were expressed inEscherichia coli and the products of both cDNAs modified the growth of ice. Transcripts of both genes accumulated late in cold acclimation in winter rye. Southern analysis of winter rye genomic DNA indicated the presence of a small gene family homologous toCHT46. In hexaploid wheat, CHT46 homologs mapped to the homeologous group 1 chromosomes and were expressed in response to cold and drought. We conclude that two novel cold-responsive genes encoding chitinases with ice-binding activity may have arisen in winter rye and other cereals through gene duplication.

Meristematic tissues from rye (Secale cereale) and oat (Avena sativa) were studied in an isothermal calorimeter at −3°C. When the frozen tissue was placed in the calorimeter, the pressure increased within 4 d to 25 and 9 kPa above ambient pressure in the sample vessels containing crowns of rye and oat, respectively. Concurrently, the thermal output went down to −194 μW in rye over the 4-d period; this negative thermal activity could be accounted for by ice melting in the plants. When the pressure was released, the output from the calorimeter went from −194 to 229 μW within 1 h, suggesting that water had frozen in the plants. We propose that CO2 from respiration had dissolved in the water in the plants and caused melting of ice (heat absorption) due to the colligative properties of solutions. When the pressure was released, the CO2 came out of solution and the water froze (heat evolution). These thermal observations were duplicated in a simplified, non-biological system using a glycol/water mixture that was partially frozen at −3°C.

During cold acclimation, winter rye (Secale cereale L.) plants secrete antifreeze proteins that are similar to pathogenesis-related (PR) proteins. In this experiment, the secretion of PR proteins was induced at warm temperatures by infection with pink snow mold (Microdochium nivale), a pathogen of overwintering cereals. A comparison of cold-induced and pathogen-induced proteins showed that PR proteins accumulated in the leaf apoplast to a greater level in response to cold. The PR proteins induced by cold and by snow mold were similar when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting. Both groups of PR proteins contained glucanase-like, chitinase-like, and thaumatin-like proteins, and both groups exhibited similar levels of glucanase and chitinase activities. However, only the PR proteins induced by cold exhibited antifreeze activity. Our findings suggest that the cold-induced PR proteins may be isoforms that function as antifreeze proteins to modify the growth of ice during freezing while also providing resistance to the growth of low-temperature pathogens in advance of infection. Both functions of the cold-induced PR proteins may improve the survival of overwintering cereals.

The lipid composition of the inner and outer membranes of the chloroplast envelope isolated from winter rye (Secale cereale L. cv Puma) leaves was characterized before and after cold acclimation. In nonacclimated leaves the inner membrane contained high proportions of monogalactosyldiacylglycerols (MGDG, 47.9 mol% of the total lipids) and digalactosyldiacylglycerols (DGDG, 31.1 mol%) and a low proportion of phosphatidylcholine (PC, 8.1 mol%). The outer membrane contained a similar proportion of DGDG (30.0 mol%); however, the proportion of MGDG was much lower (20.1 mol%) and the proportion of PC was much higher (31.5 mol%). After 4 weeks of cold acclimation, the proportions of these lipid classes were significantly altered in both of the inner and outer membranes. In the inner membrane the proportion of MGDG decreased (from 47.9 to 38.4 mol%) and the proportion of DGDG increased (from 31.1 to 39.3 mol%), with only a slight change in the proportion of PC (from 8.1 to 8.8 mol%). In the outer membrane MGDG decreased from 20.1 to 14.8 mol%, DGDG increased from 30.0 to 39.9 mol%, and PC decreased from 31.5 to 25.4 mol%. Thus, both before and after cold acclimation, the proportion of MGDG was much higher in the inner membrane than in the outer membrane. In contrast, the proportion of PC was higher in the outer membrane than in the inner membrane. The relationship between the lipid composition of the inner and outer membranes of the chloroplast envelope and freeze-induced membrane lesions is discussed.

By comparing growth under five different temperature and irradiance regimes (20[deg]C and 800, 250, and 50[mu]mol m-2 s-1 and 5[deg]C and 250 and 50 [mu]mul m-2 s-1), we have examined the effects of light, temperature, and the relative reduction state of photosystem II on plant morphology, freezing tolerance (lethal temperature at which freezing injury occurs [LT50]), transcript levels of Lhcb and two cold-stimulated genes (Wcs19 and Wcs120), and photosynthetic adjustment in winter rye (Secale cereale L. cv Musketeer). We show, for the first time to our knowledge, that in addition to adjustments in photosynthetic capacity, nonphotochemical quenching capacity and tolerance to photoinhibition, the accumulation of the cold-induced transcript Wcs19, and the compact plant morphology usually associated with cold-hardening are correlated with the relative reduction state of photosystem II rather than with growth temperature or growth irradiance per se. In contrast, the acquisition of maximal LT50, as well as Lhcb and Wcs120 mRNA accumulation, appears to be dependent on both growth temperature and growth irradiance but in an independent, additive manner. The results are discussed with respect to the possible role of the modulation of chloroplastic redox poise in photosynthetic acclimation to cold-hardening temperatures and the attainment of maximal LT50.

Several cold-regulated (COR) polypeptides, which have little or no amino acid sequence identity with known proteins, are synthesized during cold acclimation of Arabidopsis thaliana. However, the function of the polypeptides has yet to be elucidated. The objective of this study was to determine if COR6.6 and COR15am influence the incidence of either freeze-induced fusion or freeze-induced leakage of small unilamellar vesicles (SUVs) composed of either a single species of phosphatidylcholine (either 1-palmitoyl-2-oleoyl-, dioleoyl-, or dilinoleoylphosphatidylcholine), a mixture of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and free sterols (1:1:1, mol:mol), or the total lipid extract of the plasma membrane of either nonacclimated or cold-acclimated rye leaves. When the SUVs were suspended in a dilute tris(hydroxymethyl)-aminomethane/2-(N-morpholino)ethanesulfonic acid buffer, both COR6.6 and COR15am invariably decreased the incidence of freeze-induced fusion regardless of the lipid composition. However, if the SUVs were suspended in a dilute solution of either sucrose or NaCl, the COR polypeptides had little or no effect on the incidence of freeze-induced fusion. Moreover, the COR polypeptides did not decrease the incidence of freeze-induced leakage[mdash]regardless of whether the SUVs were suspended in either the dilute buffer alone or with added sucrose or NaCl. In fact, with SUVs composed of a single species of phosphatidylcholine suspended in the dilute buffer, the COR polypeptides resulted in an anomalous increase in freeze-induced leakage. When considered collectively, these results suggest that neither COR6.6 nor COR15am has a direct cryoprotective effect on SUVs frozen in vitro.

During cold acclimation, antifreeze proteins (AFPs) that are similar to pathogenesis-related proteins accumulate in the apoplast of winter rye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to modify the growth of ice. To elucidate the role of AFPs in the freezing process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from cold-acclimated rye leaves, crowns, and roots exhibited antifreeze activity, whereas no antifreeze activity was observed in extracts from nonacclimated rye plants. Antibodies raised against three apoplastic rye AFPs, corresponding to a glucanase-like protein (GLP, 32 kD), a chitinase-like protein (CLP, 35 kD), and a thaumatin-like protein (TLP, 25 kD), were used in tissue printing to show that the AFPs are localized in the epidermis and in cells surrounding intercellular spaces in cold-acclimated plants. Although GLPs, CLPs, and TLPs were present in nonacclimated plants, they were found in different locations and did not exhibit antifreeze activity, which suggests that different isoforms of pathogenesis-related proteins are produced at low temperature. The location of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. The distributions of pathogenesis-induced and cold-accumulated GLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice enter and propagate through plant tissues.

The ability to control extracellular ice formation during freezing is critical to the survival of freezing-tolerant plants. Antifreeze proteins, which are proteins that have the ability to retard ice crystal growth, were recently identified as the most abundant apoplastic proteins in cold-acclimated winter rye (Secale cereale L.) leaves. In the experiments reported here, amino-terminal sequence comparisons, immuno-cross-reactions, and enzyme activity assays all indicated that these antifreeze proteins are similar to members of three classes of pathogenesis-related proteins, namely, endochitinases, endo-beta-1,3-glucanases, and thaumatin-like proteins. Apoplastic endochitinases and endo-beta-1,3-glucanases that were induced by pathogens in freezing-sensitive tobacco did not exhibit antifreeze activity. Our findings suggest that subtle structural differences may have evolved in the pathogenesis-related proteins that accumulate at cold temperatures in winter rye to confer upon these proteins the ability to bind to ice.

The effect of a short-term (hours) shift to low temperature (5[deg]C) and long-term (months) cold hardening on photosynthesis and carbon metabolism was studied in winter rye (Secale cereale L. cv Musketeer). Cold-hardened plants grown at 5[deg]C exhibited 25% higher in situ CO2 exchange rates than nonhardened plants grown at 24[deg]C. Cold-hardened plants maintained these high rates throughout the day, in contrast to nonhardened plants, which showed a gradual decline in photosynthesis after 3 h. Associated with the increase in photosynthetic capacity following cold hardening was an increase in ribulose-1,5-bisphosphate carboxylase/oxygenase and sucrose phosphate synthase activity and 3- to 4-fold increases in the pools of associated metabolites. Leaves of nonhardened plants shifted overnight to 5[deg]C required 9 h in the light at 5[deg]C before maximum rates of photosynthesis were reached. The gradual increase in photosynthesis in leaves shifted to 5[deg]C was correlated with a sharp decline in the 3-phosphoglycerate/triose phosphate ratio and by an increase in the ribulose bisphosphate/3-phosphoglycerate ratio, indicating the gradual easing of aninorganic phosphate-mediated feedback inhibition on photo-synthesis. We suggest that the strong recovery of photosynthesis in winter rye following cold hardening indicates that the buildup of photosynthetic enzymes, as well as those involved in sucrose synthesis, is an adaptive response that enables these plants to maximize the production of sugars that have both cryoprotective and storage functions that are critical to the performance of these cultivars during over-wintering.

There have been very few reports on the expression of stress-responsive genes in field-grown material. A barley dehydrin cDNA was used to investigate the expression of dehydrin-like transcripts after low-temperature and abscisic acid-induced acclimation of bromegrass (Bromus inermis Leyss) suspension cells and of bromegrass and rye (Secale cereale) plants grown in the field and under controlled environmental conditions. Field-acclimated plants accumulated high levels of dehydrin transcripts and were very freezing tolerant. Plants grown in pots and hydroponics under controlled environments also accumulated dehydrin transcripts and showed increased freezing tolerance. Simulation of a combined drought and freezing stress in pots resulted in expression of dehydrin-like transcripts comparable to those observed in field-acclimated material.

Apoplastic extracts of cold-acclimated winter rye (Secale cereale L. cv Musketeer) leaves were previously shown to exhibit antifreeze activity. The objectives of the present study were to identify and characterize individual antifreeze proteins present in the apoplastic extracts. The highest protein concentrations and antifreeze activity were obtained when the leaf apoplast was extracted with ascorbic acid and either CaCl2 or MgSO4. Seven major polypeptides were purified from these extracts by one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under nonreducing conditions. The five larger polypeptides, of 19, 26, 32, 34, and 36 kD, exhibited significant levels of antifreeze activity, whereas the 11- and 13-kD polypeptides showed only weak activity. Five of these polypeptides migrated with higher apparent molecular masses on SDS gels after treatment with 0.1 M dithiothreitol, which indicated the presence of intramolecular disulfide bonds. The apparent reduction of the disulfide bonds did not eliminate antifreeze activity in four of the polypeptides that contained intramolecular disulfide bonds and exhibited significant levels of antifreeze activity. The amino acid compositions of these polypeptides were similar in that they were all relatively enriched in the residues Asp/Asn, Glu/Gln, Ser, Thr, Gly, and Ala; they all lacked His, except for the 26-kD polypeptide, and they contained up to 5% Cys residues. These polypeptides were examined with antisera to other cystine-containing antifreeze proteins from fish and insects, and no common epitopes were detected. We conclude that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifreezes produced by fish and insects.

Freezing injury in protoplasts isolated from leaves of nonaccli-mated rye (Secale cereale cv Puma) is associated with the formation of the inverted hexagonal (HII) phase. However, in protoplasts from cold-acclimated rye, injury is associated with the occurrence of localized deviations in the fracture plane, a lesion referred to as the "fracture-jump lesion." To establish that these ultrastructural consequences of freezing are not unique to protoplasts, we have examined the manifestations of freezing injury in leaves of non-acclimated and cold-acclimated rye by freeze-fracture electron microscopy. At -10[deg]C, injury in nonacclimated leaves was manifested by the appearance of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and by the frequent occurrence of the HII phase. The HII phase was not observed in leaves of cold-acclimated rye frozen to -35[deg]C. Rather, injury was associated with the occurrence of the fracture-jump lesion between the plasma membrane and closely appressed cytoplasmic membranes. Studies of the time dependence of HII phase formation in nonacclimated leaves indicated that freeze-induced dehydration requires longer times in leaves than in isolated protoplasts. These results demonstrate that the freeze-induced formation of the HII phase in nonacclimated rye and the fracture-jump lesion in cold-acclimated rye are not unique to protoplasts but also occur in the leaves from which the protoplasts are isolated.

Winter cultivars of rye (Secale cereale L., cv Musketeer) and wheat (Triticum aestivum L. cvs Kharkov and Monopol), but not a spring cultivar of wheat (Glenlea), grown at cold-hardening temperatures showed, at high irradiances, a higher proportion of oxidized to reduced primary, stable quinone receptor (QA) than did the same cultivars grown under nonhardening conditions. In addition, there was a positive correlation between the effects of low-growth temperature on this increased proportion of oxidized QA, and a concomitant increase in the capacity for photosynthesis, and LT50, the temperature at which 50% of the seedlings are killed, in cultivars showing different freezing tolerances. This suggests that low-temperature modulation of the photosynthetic apparatus may be an important factor during the induction of freezing resistance in cereals. Finally, the control of photosystem II photochemistry by nonphotochemical quenching of excitation energy was identical for nonhardened and cold-hardened winter rye. However, examination of measuring temperature effects per se revealed that, irrespective of growth temperature, nonphotochemical quenching exerted a stronger control on photosystem II photochemistry at 10[deg] C rather than at 20[deg] C.

The enzyme catalase (EC 1.11.1.6) is light sensitive and subject to a rapid turnover in light, similar to the D1 reaction center protein of photosystem II. After 3 h of preadaptation to darkness or to different light intensities (90 and 520 μmol m−2 s−1 photosynthetic photon flux density), sections of rye leaves (Secale cereale L.) were labeled for 4 h with l-[35S]methionine. From leaf extracts, catalase was immunoprecipitated with an antiserum prepared against the purified enzyme from rye leaves. Both incorporation into catalase and degradation of the enzyme polypeptide during a subsequent 16-h chase period increased with light intensity. At a photon flux density of 520 μmol m−2 s−1, the apparent half-time of catalase in rye leaves was 3 to 4 h, whereas that of the D1 protein was much shorter, about 1.5 h. Exposure to stress conditions, such as 0.6 m NaCl or a heat-shock temperature of 40°C, greatly suppressed both total protein synthesis and incorporation of the label into catalase and into the D1 protein. Immunoblotting assays indicated that in light, but not in darkness, steady-state levels of catalase and of the D1 protein strongly declined during treatments with salt, heat shock, or translation inhibitors that block repair synthesis. Because of the common property of rapid photodegradation and the resulting dependence on continuous repair, declines in catalase as well as of the D1 protein represent specific and sensitive indicators for stress conditions that suppress the translational activities of 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.

The role of acyl lipids in the in vitro stabilization of the oligomeric form of light-harvesting complex II of winter rye (Secale cereale L. cv Muskateer) grown at 5 or 20 degrees C was investigated. Purified light-harvesting complex II was enzymically delipidated to various extents by treatment with the following lipolytic enzymes: phospholipase C, phospholipase A(2), and galactolipase. Complete removal of phosphatidylcholine had no effect on the stability of the oligomeric form, whereas the removal of phosphatidylcholine plus phosphatidylglycerol caused a decrease in the ratio of oligomeric:monomeric forms from 1.86 +/- 0.17 to 0.85 +/- 0.17 and 3.51 +/- 0.82 to 0.81 +/- 0.29 for purified cold-hardened and nonhardened light-harvesting complex II, respectively, with no change in free pigment content. Incubation of delipidated cold-hardened or nonhardened light-harvesting complex with purified thylakoid phosphatidylglycerol containing trans-Delta(3)-hexadecenoic acid resulted in 48% reconstitution of the oligomeric form on a total chlorophyll basis with an oligomer:monomer of about 1.90. Incubation in the presence of di- 16:0 or di- 18:1 phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglyceride, or digalactosyldiacylglyceride caused no oligomerization, but rather a further destabilization of the monomeric form. These lipid-dependent structural changes were correlated with significant changes in the 77K fluorescence emission spectra for purified light-harvesting complex II. We conclude that the stabilization of the supramolecular organization of light-harvesting complex II from rye is specifically dependent upon molecular species of phosphatidylglycerol containing trans-Delta(3)-hexadecenoic acid.

A survey of isolated thylakoids from 11 different higher plant species (Spinacia oleracea L., Pisum sativum L., Vicia faba L., Brassica napus L., Vigna sinensis L., Vinca minor L., Secale cereale L., Triticum aestivum L., Triticosecale Wittn., Hordeum vulgare L., Zea mays L.) indicated that the ratio of the oligomeric:monomeric form of the light-harvesting complex II was twofold higher for the dicots (3.16 ± 0.35) than the monocots (1.64 ± 0.25) examined under identical separation procedures. Under conditions specifically designed to stabilize the oligomeric form in vitro, we show that the oligomeric form of dicot light-harvesting complex II is twice as stable to solubilization in the presence of sodium dodecyl sulfate (SDS) than that observed for monocots. This decreased stability of monocot light-harvesting complex II is associated with a twofold increase in the trienoic fatty acid level of thylakoid phosphatidylglycerol but with no significant changes in the trienoic fatty acid levels in the major galactolipids. In addition, SDS polyacrylamide gel electrophoresis and western blot analyses with monoclonal antibodies indicated that monocots exhibited greater heterogeneity in the polypeptide complements associated with subfractions of light-harvesting complex II than the dicots examined. The data indicate that the oligomeric form of the light-harvesting complex II is not the result of a simple oligomerization of a common monomeric unit. We suggest that the difference in stability of the oligomeric form of light-harvesting complex II in isolated thylakoids of monocots and dicots is probably due to a differential accessibility to SDS. The differential SDS accessibility may be due to differences in thylakoid protein-protein and/or protein-lipid interactions.

During cold acclimation of Puma rye (Secale cereale L. cv Puma), the intracellular osmotic potential nearly doubles. During this period, the accumulation of glycinebetaine, proline, and soluble sugars was monitored. The amount of glycinebetaine increased from 290 to 1300 micrograms per gram fresh weight during the 4-week acclimation period. Proline content did not change during the first 3 weeks of acclimation but then increased from 27 to 580 micrograms per gram fresh weight during the next 3 weeks. The total soluble sugar content more than doubled by the second week of cold acclimation, increasing from 11 to 26 milligrams per gram fresh weight. Most of this increase can be attributed to the accumulation of sucrose and raffinose, whose levels increased from 2.4 and 0 to 11 and 5 milligrams per gram fresh weight, respectively. The content of monosaccharides, predominantly glucose, remained at a constant 10 milligrams per gram fresh weight throughout the acclimation period. A comparison of the sugar content of protoplasts versus vacuoles isolated from cold-acclimated leaves revealed that the extravacuolar volume contained monosaccharides, sucrose, and raffinose. Thus, the increased amounts of sucrose and raffinose that occur during cold acclimation are present in compartments external to the vacuole and may contribute to cryoprotection.

We have examined the occurrence/disappearance, tissue location, and posttranslational modification of beta-amylase proteins in rye (Secale cereale L.) kernels at three physiological stages (development, maturity, germination) with a normal inbred line and a mutant line exhibiting a high but incomplete beta-amylase deficiency. This deficiency corresponds to a lack of accumulation of beta-amylase activity in the endosperm and does not affect the level of activity in the outer pericarp and green tissues as compared to the normal line. Two antigenically related but distinct beta-amylases (I and II) were detected in the normal line (II being the major constituent) and only one (I) in the mutant line. I and II display very similar electrophoretic polymorphism. In both lines, I appears to be ubiquitous, although it disappears from the outer pericarp during ripening. Antigen II was present only in the normal line and appears to be specific for the endosperm and perhaps for the maternal green tissues of the seed. Posttranslational modifications occurring during germination, which are mimicked by the action of papain, affect II but not I. The two groups of beta-amylases are discussed in relation to recent reports indicating the presence of two types of beta-amylase with different functions and gene loci in barley and wheat.

The growth kinetics of spinach plants (Spinacia oleracea L. cv Savoy) grown at 5°C or 16°C were determined to allow us to compare leaf tissues of the same developmental stage rather than chronological age. The second leaf pairs reached full expansion at a plant age of 32 and 92 days for the 16°C and 5°C plants, respectively. Growth at 5°C resulted in an increased leaf area, dry weight, dry weight per area, and leaf thickness. Despite these changes, pigment content and composition, room temperature in vivo fluorescence, and apparent quantum yield and light-saturated rates of CO2 exchange or O2 evolution were not affected by the growth temperature. Furthermore, 5°C expanded leaves were found to be more resistant to photoinhibition at 5°C than were 16°C expanded leaves. Thus, it is concluded that spinach grown at low temperature is not stressed. However, shifting spinach leaves from 5°C to 16°C or from 16°C to 5°C for 12 days after full leaf expansion had occurred resulted in a 20 to 25% reduction in apparent quantum yields and 50 to 60% reduction in light saturated rates of both CO2 exchange and O2 evolution. This was not accompanied by a change in the pigment content or composition or in the room temperature in vivo fluorescence. It appears that leaf aging during the temperature shift period can account for the reduction in photosynthesis. Comparison of cold-hardened and non-hardened winter rye (Secale cereale L. cv Muskateer) with spinach by in vivo fluorescence indicated that rye is more sensitive to both short term and longer duration temperature shifts than is spinach. Thus, susceptibility to an abrupt temperature shift appears to be species dependent.

The cold stability of cortical microtubules in root-tip cells of winter rye (Secale cereale L. cv Puma) is altered by growth temperature (GP Kerr, JV Carter [1990] Plant Physiol 93:77-82). One hypothesis for the basis of this alteration is that different tubulin isotypes are present at different growth temperatures, and that the cold stability of microtubules is affected by these isotypic differences. We have explored the first part of this hypothesis by comparing protein extracts from roots of seedlings grown for 2 days at 22 degrees C (nonacclimated) or for an additional 2 or 4 days at 4 degrees C (cold-acclimated). Immunoblots of two-dimensional polyacrylamide gels were probed with monoclonal antibodies to alpha- and beta-tubulin. At least six alpha- and seven beta-tubulins were present in the extracts from both the nonacclimated and cold-acclimated roots. Changes in electrophoretic mobility and isotype number of both alpha- and beta-tubulin were observed after only 2 days at 4 degrees C. Further changes in tubulin were observed after 4 days at 4 degrees C. Changes in alpha-tubulin were more pronounced than those in beta-tubulin.

The response of cortical microtubules to low temperature and freezing was assessed for root tips of cold-acclimated and non-acclimated winter rye (Secale cereale L. cv Puma) seedlings using indirect immunofluorescence microscopy with antitubulin antibodies. Roots cooled to 0 or -3 degrees C were fixed for immunofluorescence microscopy at these temperatures or after an additional hour at 4 degrees C. Typical arrays of cortical microtubules were present in root-tip cells of seedlings exposed to the cold-acclimation treatment of 4 degrees C for 2 days. Microtubules in these cold-acclimated cells were more easily depolymerized by a 0 degrees C treatment than microtubules in root-tip cells of nonacclimated, 22 degrees C-grown seedlings. Microtubules were still present in some cells of both nonacclimated and cold-acclimated roots at 0 and -3 degrees C; however, the number of microtubules in these cells was lower than in controls. Microtubules remaining during the -3 degrees C freeze were shorter than microtubules in unfrozen control cells. Repolymerization of microtubules after both the 0 and -3 degrees C treatments occurred within 1 h. Root tips of nonacclimated seedlings had an LT-50 of -9 degrees C. Cold acclimation lowered this value to -14 degrees C. Treatment of 22 degrees C-grown seedlings for 24 h with the microtubule-stabilizing drug taxol caused a decrease in the freezing tolerance of root tips, indicated by a LT-50 of -3 degrees C. Treatment with D-secotaxol, an analog of taxol that was less effective in stabilizing microtubules, did not alter the freezing tolerance. We interpret these data to indicate that a degree of depolymerization of microtubules is necessary for realization of maximum freezing tolerance in root-tip cells of rye.

A procedure has been developed for the vitrification of mesophyll protoplasts isolated from leaves of nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings (Secale cereale L. cv Puma). The procedure involves (a) equilibration (loading) of the protoplasts with an intermediate concentration (1.5, 1.75, or 2.0 molar) of ethylene glycol (EG) at 20 degrees C; (b) dehydration of the protoplasts in a concentrated vitrification solution made of 7 molar EG + 0.88 molar sorbitol + 6% (w/v) bovine serum albumin (BSA) at 0 degrees C; (c) placing the protoplasts into polypropylene straws and quenching in liquid nitrogen (LN(2)); and (d) recovery of the protoplasts from LN(2) and removal (unloading) of the vitrification solution. For NA protoplasts, 47 + 1% survival was obtained following recovery from LN(2) if the protoplasts were first loaded with 1.75 molar EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a hypertonic (2.0 osmolal [osm]) sorbitol solution. If they were unloaded in an isotonic solution (0.53 osm), survival was 3+/-2%. In contrast, survival of ACC protoplasts following recovery from LN(2) was 34 +/- 10% when the protoplasts were loaded in a 2.0 molar EG solution and unloaded in an isotonic sorbitol solution (1.03 osm). If ACC protoplasts were unloaded in an hypertonic sorbitol solution (1.5 osm), survival was 51 +/- 9%. These results indicate that the osmotic excursions incurred during the procedure are a major factor affecting survival.

Many plants increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Cold acclimation in Arabidopsis involves rapid cold-induced expression of the C-repeat/dehydration-responsive element binding factor (CBF) transcriptional activators followed by expression of CBF-targeted genes that increase freezing tolerance. Here, we present evidence for a CBF cold-response pathway in Brassica napus. We show that B. napus encodes CBF-like genes and that transcripts for these genes accumulate rapidly in response to low temperature followed closely by expression of the cold-regulated Bn115 gene, an ortholog of the Arabidopsis CBF-targeted COR15a gene. Moreover, we show that constitutive overexpression of the Arabidopsis CBF genes in transgenic B. napus plants induces expression of orthologs of Arabidopsis CBF-targeted genes and increases the freezing tolerance of both nonacclimated and cold-acclimated plants. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat (Triticum aestivum L. cv Norstar) and rye (Secale cereale L. cv Puma), which cold acclimate, as well as in tomato (Lycopersicon esculentum var. Bonny Best, Castle Mart, Micro-Tom, and D Huang), a freezing-sensitive plant that does not cold acclimate. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved amino acid sequences, PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. We conclude that components of the CBF cold-response pathway are highly conserved in flowering plants and not limited to those that cold acclimate.

Antifreeze activity is induced by cold temperatures in winter rye (Secale cereale) leaves. The activity arises from six antifreeze proteins that accumulate in the apoplast of winter rye leaves during cold acclimation. The individual antifreeze proteins are similar to pathogenesis-related proteins, including glucanases, chitinases, and thaumatin-like proteins. The objective of this study was to study the regulation of antifreeze activity in response to ethylene and salicyclic acid, which are known regulators of pathogenesis-related proteins induced by pathogens. Nonacclimated plants treated with salicylic acid accumulated apoplastic proteins with no antifreeze activity. In contrast, when nonacclimated plants were exposed to ethylene, both antifreeze activity and the concentration of apoplastic protein increased in rye leaves. Immunoblotting revealed that six of the seven accumulated apoplastic proteins consisted of two glucanases, two chitinases, and two thaumatin-like proteins. The ethylene-releasing agent ethephon and the ethylene precursor 1-aminocyclopropane-1-carboxylate also induced high levels of antifreeze activity at 20°C, and this effect could be blocked by the ethylene inhibitor AgNO3. When intact rye plants were exposed to 5°C, endogenous ethylene production and antifreeze activity were detected within 12 and 48 h of exposure to cold, respectively. Rye plants exposed to drought produced both ethylene and antifreeze activity within 24 h. We conclude that ethylene is involved in regulating antifreeze activity in winter rye in response to cold and drought.

Al-Induced secretion of organic acids from the roots has been considered as a mechanism of Al tolerance, but the processes leading to the secretion of organic acids are still unknown. In this study, the secretion pattern and alteration in the metabolism of organic acids under Al stress were examined in rye (Secale cereale L. cv King) and wheat (Triticum aestivum L. cv Atlas 66). Al induced rapid secretion of malate in the wheat, but a lag (6 and 10 h for malic and citric acids, respectively) between the exposure to Al and the secretion of organic acids was observed in the rye. The activities of isocitrate dehydrogenase, phosphoenolpyruvate carboxylase, and malate dehydrogenase were not affected by Al in either plant. The activity of citrate synthase was increased by the exposure to Al in the rye, but not in the wheat. The secretion of malate was not suppressed at low temperature in the wheat, but that of citrate was stopped in the rye. The Al-induced secretion of citrate from roots of the rye was inhibited by the inhibitors of a citrate carrier, which transports citrate from the mitochondria to the cytoplasm. All of these results suggest that alteration in the metabolism of organic acids is involved in the Al-induced secretion of organic acids in rye, but only activation of an anion channel seems to be responsible for the rapid secretion of malate in the wheat.

An endogenous wheat (Triticum aestivum) flour endoxylanase was purified to homogeneity from a crude wheat flour extract by ammonium sulfate precipitation and cation-exchange chromatography. The 30-kD protein had an isoelectric point of 9.3 or higher. A sequence of 19 amino acids at the NH2 terminus showed 84.2% identity with an internal sequence of the 15-kD grain-softness protein, friabilin. High-performance anion-exchange chromatography and gel-permeation analysis of the hydrolysis products indicated the preferential hydrolysis of highly branched structures by the enzyme; wheat arabinoxylan and rye (Secale cereale) arabinoxylan (high arabinose to xylose ratios) were hydrolyzed more efficiently by this enzyme than oat (Avena sativa) spelt xylan (low arabinose to xylose ratios). The release of the hydrolysis products as a function of time suggested that the endoxylanolytic activity was associated with the release of arabinose units from the polysaccharides, suggesting that the enzyme action is similar to that by endoxylanases from Ceratocystis paradoxa, Aspergillus niger, and Neurospora crassa. Although the enzyme released arabinose from arabinoxylan, it did not hydrolyze p-nitrophenyl-α--L-arabinofuranoside. From the above, it follows that the enzyme, called arabinoxylanase, differs from most microbial endoxylanases and from an endoxylanase purified earlier from wheat flour.

In the dark, all decarboxylation reactions are associated with the oxidase reactions of mitochondrial electron transport. In the light, photorespiration is also active in photosynthetic cells. In winter rye (Secale cereale L.), cold hardening resulted in a 2-fold increase in the rate of dark respiratory CO2 release from leaves compared with nonhardened (NH) controls. However, in the light, NH and cold-hardened (CH) leaves had comparable rates of oxidase decarboxylation and total intracellular decarboxylation. Furthermore, whereas CH leaves showed similar rates of total oxidase decarboxylation in the dark and light, NH leaves showed a 2-fold increase in total oxidase activity in the light compared with the dark. Light suppressed oxidase decarboxylation of end products of photosynthesis 2-fold in NH leaves and 3-fold in CH leaves in air. However, in high-CO2, light did not suppress the oxidase decarboxylation of end products. Thus, the decrease in oxidase decarboxylation of end products observed in the light and in air reflected glycolate-cycle-related inhibition of tricarboxylic acid cycle activity. We also showed that the glycolate cycle was involved in the decarboxylation of the end products of photosynthesis in both NH and CH leaves, suggesting a flow of fixed carbon out of the starch pool in the light.

Extracellular ice formation in frost-tolerant organisms is often initiated at specific sites by ice nucleators. In this study, we examined ice nucleation activity (INA) in the frost-tolerant plant winter rye (Secale cereale). Plants were grown at 20[deg]C, at 5[deg]C with a long day, and at 5[deg]C with a short day (5[deg]C-SD). The threshold temperature for INA was -5 to -12[deg]C in winter rye leaves from all three growth treatments. Epiphytic ice nucleation-active bacteria could not account for INA observed in the leaves. Therefore, the INA must have been produced endogenously. Intrinsic rye ice nucleators were quantified and characterized using single mesophyll cell suspensions obtained by pectolytic degradation of the leaves. The most active ice nucleators in mesophyll cell suspensions exhibited a threshold ice nucleation temperature of -7[deg]C and occurred infrequently at the rate of one nucleator per 105 cells. Rye cells were treated with chemicals and enzymes to characterize the ice nucleators, which proved to be complexes of proteins, carbohydrates, and phospholipids, in which both disulfide bonds and free sulfhydryl groups were important for activity. Carbohydrates and phospholipids were important components of ice nucleators derived from 20[deg]C leaves, whereas the protein component was more important in 5[deg]C-SD leaves. This difference in composition or structure of the ice nucleators, combined with a tendency for more frequent INA, suggests that more ice nucleators are produced in 5[deg]C-SD leaves. These additional ice nucleators may be a component of the mechanism for freezing tolerance observed in winter rye.

Ethylene evolution from etiolated barley (Hordeum vulgare), wheat (Triticum aestivum), and rye (Secale cereale) seedlings during coleoptile growth followed a rhythmic pattern, with a period of about 16 h for barley and wheat and 12 h for rye seedlings. Leaf emergence disturbed the established rhythm of ethylene evolution.

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5 degrees C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20 degrees C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [(14)C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.

Thylakoids isolated from winter rye (Secale cereale L. cv Muskateer) grown at 5 degrees C or 20 degrees C were compared with respect to their capacity to exhibit an increase in light saturated rates of photosystem I (PSI) electron transport (ascorbate/dichlorophenolindophenol --methylviologen) after dark preincubation at temperatures between 0 and 60 degrees C. Thylakoids isolated in the presence or absence of Na(+)/Mg(2+) from 20 degrees C grown rye exhibited transient, 40 to 60% increases in light saturated rates of PSI activity at all preincubation temperatures between 5 and 60 degrees C. This increase in PSI activity appeared to occur independently of the electron donor employed. The capacity to exhibit this in vitro induced increase in PSI activity was examined during biogenesis of rye thylakoids under intermittent light conditions at 20 degrees C. Only after exposure to 48 cycles (1 cycle = 118 minutes dark + 2 min light) of intermittent light did rye thylakoids exhibit an increase in light saturated rates of PSI activity even though PSI activity could be detected after 24 cycles. In contrast to thylakoids from 20 degrees C grown rye, thylakoids isolated from 5 degrees C grown rye in the presence of Na(+)/Mg(2+) exhibited no increase in light saturated PSI activity after preincubation at any temperature between 0 and 60 degrees C. This was not due to damage to PSI electron transport in thylakoids isolated from 5 degrees C grown plants since light saturated PSI activity was 60% higher in 5 degrees C thylakoids than 20 degrees C thylakoids prior to in vitro dark preincubation. However, a two-fold increase in light saturated PSI activity of 5 degrees C thylakoids could be observed after dark preincubation only when 5 degrees C thylakoids were initially isolated in the absence of Na(+)/Mg(2+). We suggest that 5 degrees C rye thylakoids, isolated in the presence of these cations, exhibit light saturated PSI electron transport which may be closer to the maximum rate attainable in vitro than 20 degrees C thylakoids and hence cannot be increased further by dark preincubation.

Both small and large sizes of phytochrome purified from Garry oat (Avena sativa L. ev. Garry) as well as large phytochrome purified from Newton oat (A. sativa L. cv. Newton), rye (Secale cereale L. cv. Balbo), barley (Hordeum vulgare L. cv. Harrison), and pea (Pisum sativum L. cv. Alaska) seedlings are characterized by a specific antiserum against large Garry oat phytochrome. A spur is observed by double diffusion assay against large and small Garry oat phytochrome indicating only partial identity. In micro-complement fixation assays, large Garry oat phytochrome yields greater activity than small Garry oat phytochrome. In addition, the peak of activity is shifted to a higher antigen concentration with small phytochrome. Phytochrome, red-absorbing form, and phytochrome, far redabsorbing form, are indistinguishable by both double diffusion and micro-complement fixation assay. The different grass phytochromes are antigenically identical by double diffusion assay. Immunoelectrophoretic analyses of oat and rye large phytochrome, after proteolysis, suggest that there are one or a few regions of the molecule especially susceptible to hydrolysis by a wide variety of endopeptidases.

The properties of the leucine transport systems of cells isolated from dark-grown cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) epicotyls were remarkably similar. After 1 hour of incubation, leucine was accumulated in the cells 80- to 100-fold above that of the external medium, but the transported leucine was not metabolized. Approximately one-third of the accumulated leucine was present in the vacuole after 40 minutes of incubation. At 25 degrees C, efflux of leucine from the vacuole was 6 to 10 times slower than it was from the cytoplasm, while at 5 degrees C efflux from the cells was inhibited.The apparent K(m) and V(max) for leucine uptake for both types of cells were of the order of 20 to 60 micromolar and 0.5 to 1.3 nanomoles per minute per 10(6) cells. The pH and temperature optima for both types of cells were 5 and 25 degrees C, respectively. The leucine transport system for these cells was relatively specific for amino acids lacking either bulky or charged groups on the amino acid side chains.Arrhenius plots for leucine uptake by hardened and nonhardened cells showed discontinuities at 13 degrees C, and the energies of activation were similar. The results suggests that biochemical changes which occur in rye cells upon cold hardening did not result in an observable perturbation of the properties of the leucine transport system.

Highly enriched plasma membrane fractions were isolated from leaves of nonacclimated (NA) and acclimated (ACC) rye (Secale cereale L. cv Puma) seedlings. Collectively, free sterols, steryl glucosides, and acylated steryl glucosides constituted >50 mole% of the total lipid in both NA and ACC plasma membrane fractions. Glucocerebrosides containing hydroxy fatty acids constituted the major glycolipid class of the plasma membrane, accounting for 16 mole% of the total lipid. Phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and phosphatidylinositol, comprised only 32 mole% of the total lipid in NA samples. Following cold acclimation, free sterols increased from 33 to 44 mole%, while steryl glucosides and acylated steryl glucosides decreased from 15 to 6 mole% and 4 to 1 mole%, respectively. Sterol analyses of these lipid classes demonstrated that free beta-sitosterol increased from 21 to 32 mole% (accounting for the increase in free sterols as a class) at the expense of sterol derivatives containing beta-sitosterol. Glucocerebrosides decreased from 16 to 7 mole% of the total lipid following cold acclimation. In addition, the relative proportions of associated hydroxy fatty acids, including 22:0 (h), 24:0 (h), 22:1 (h), and 24:1 (h), were altered. The phospholipid content of the plasma membrane fraction increased to 42 mole% of the total lipid following cold acclimation. Although the relative proportions of the individual phospholipids did not change appreciably after cold acclimation, there were substantial differences in the molecular species. Di-unsaturated molecular species (18:2/18:2, 18:2/18:3, 18:3/18:3) of phosphatidylcholine and phosphatidylethanolamine increased following acclimation. These results demonstrate that cold acclimation results in substantial changes in the lipid composition of the plasma membrane.

Preparation and characterization of antisera against lettuce (Lactuca sativa L., cv. Grand Rapids) and pea (Pisum sativum L., cv. Alaska) phytochrome is described. These antisera, together with previously obtained antisera against zucchini (Cucurbita pepo L., cv. Black Beauty) and oat (Avena sativa L., cv. Garry) phytochrome, were used to compare by Ouchterlony double immunodiffusion phytochrome isolated from etiolated lettuce, pea, bean (Phaseolus vulgaris L., cv. Taylor Horticultural Bush), zucchini, oat and rye (Secale cereale L., cv. Balbo) seedlings. Cross reactivity between monocotyledonous phytochrome and antidicotyledonous-phytochrome serum and between dicotyledonous phytochrome and antimonocotyledonous-phytochrome serum was always weak or not perceptible by this assay. Among the four dicotyledonous phytochromes examined, pea and bean were the most similar immunochemically as anticipated. Pea and lettuce phytochrome somewhat unexpectedly also exhibited similar immunochemical reactivity. Zucchini phytochrome by contrast was immunochemically distinct from pea, bean, and lettuce phytochrome, although it did react with all three antidicotyledonous-phytochrome sera. Initial attempts to identify immunoglobulins that would recognize phytochrome regardless of its source indicated that they may exist. Such immunoglobulins are of interest because they might react with one or more determinants that could be part of an active site of phytochrome. These immunoglobulins, once isolated, could thus serve as a potential probe for the active site of phytochrome.

Using plasma membranes with high purity isolated from winter rye seedlings ( Secale cereale L. cv Puma) by an aqueous two-polymer phase partition technique, lipid and protein changes were determined during the development of cold hardiness. The degree of fatty acid unsaturation and proportions of phospholipid classes changed only slightly during cold acclimation. A small change, however, was observed in sterol composition; β-sitosterol increased gradually with a corresponding decrease in campesterol plus stigmasterol during cold acclimation. The phospholipid to protein ratio increased proportionally to the increase in cold hardiness. During the period of acclimation, the sterol to phospholipid ratio declined and was inversely related to the development of cold hardiness. A distinct difference between polypeptide components of nonacclimated and acclimated plasma membrane was observed on two-dimensional sodium dodecyl sulfate slab polyacrylamide gels. A change was also detected in glycopeptides. These results suggest that the plasma membrane is in a dynamic state during cold acclimation, changing in response to the development of cold hardiness.