Structural Changes in Thylakoid Proteins during Cold Acclimation and Freezing of Winter Rye (Secale cereale L. cv. Puma)

Thylakoids were isolated from nonhardened and cold-hardened winter rye (Secale cereale L. cv. Puma), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of sulfhydryl reagents. Electrophoresis of cold-hardened rye thylakoid proteins revealed the presence of a 35 kilodalton polypeptide and the absence of a 51 kilodalton polypeptide found in nonhardened rye thylakoid proteins. The 35 kilodalton band could be induced by adding β-mercaptoethanol to nonhardened rye thylakoid proteins, whereas the 51 kilodalton band could be formed by adding cupric phenanthroline to these same proteins. Sulfhydryl group titration showed that cold-hardened rye thylakoid proteins contained more free sulfhydryls than nonhardened rye proteins. Although amino acid analysis of thylakoid proteins revealed quantitative differences in several amino acid residues, the polarity of thylakoid proteins did not change during cold acclimation. No significant changes in sodium dodecyl sulfate-polyacrylamide gels of thylakoid proteins appeared when either nonhardened or cold-hardened plants were frozen in vivo or in vitro. However, thylakoid proteins did aggregate when frozen in the presence of β-mercaptoethanol. Although thylakoid proteins isolated from cold-hardened rye contained more reduced thiols, a general state of reduction did not act as a cryoprotectant. It is hypothesized that conformational changes of specific proteins may be important for low temperature growth of rye.     

Involvement of Plasma Membrane Alterations in Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma)

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.     

Analysis of Glucocerebrosides of Rye (Secale cereale L. cv Puma) Leaf and Plasma Membrane

Glucocerebrosides of whole rye (Secale cerale L. cv Puma) leaf and plasma membrane were analyzed using gas chromatography-mass spectrometry and gas chromatography following hydrolysis or as intact molecules purified by reverse-phase high performance liquid chromatography. Fatty acids of acid-hydrolyzed leaf and plasma membrane glucocerebrosides consisted of >98 weight percent saturated and monounsaturated 2-hydroxy fatty acids which contained 16 to 26 carbon atoms. The major fatty acids detected were 2-hydroxynervonic acid (24:1h), 2-hydroxylignoceric acid (24:0h), 2-hydroxyerucic acid (22:1h), and 2-hydroxybehenic acid (22:0h). Long-chain bases of alkaline-hydrolyzed glucocerebrosides consisted primarily of cis-trans isomers of the trihydroxy base 4-hydroxysphingenine (t18:1) and the dihydroxy base sphingadienine (d18:2) with lesser amounts of 4-hydroxysphinganine (t18:0) and isomers of sphingenine (d18:1). Intact, underivatized glucocerebroside molecular species of rye leaf and plasma membrane were separated into more than 30 molecular species using reverse-phase HPLC. The molecular species composition of leaf and plasma membrane were quantitatively and qualitatively similar. The major molecular species was 24:1h-t18:1 which constituted nearly 40 weight percent of leaf and plasma membrane extracts. Several other species including 22:1h-t18:1, 24:1h-t18:1 (isomer), 22:0h-t18:1, 24:1h-d18:2, and 24:0h-t18:1 each comprised 4 to 8% of the total. It is anticipated that the high performance liquid chromatography procedure developed in this study to separate intact, underivatized lipid molecular species will be useful in future studies of the physical properties and biosynthesis of plant glucocerebrosides.     

Plasma Membrane Lipid Alterations Associated with Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma)

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 β-sitosterol increased from 21 to 32 mole% (accounting for the increase in free sterols as a class) at the expense of sterol derivatives containing β-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.     

Isolation and Identification of Plasma Membrane from Light-Grown Winter Rye Seedlings (Secale cereale L. cv Puma)

An effective method for the isolation of plasma membrane from light-grown winter rye seedlings (Secale cereale L. cv Puma) was established using a liquid two-polymer phase separation. The conditions for the specific partition of plasma membrane into the polyethylene glycol-enriched upper phase were examined, including variations in the polymer concentration, buffer system, pH, and NaCl addition in the phase partition system. The most effective phase partition system for the isolation of plasma membrane from winter rye consisted of 5.6/5.6% (w/w) polyethylene glycol 4000/dextran T500 in 0.25 molar sucrose-10 millimolar potassium phosphate-30 millimolar NaCl (pH 7.8), repeated once. When the isolated plasma membrane was centrifuged on a linear sucrose density gradient, a single band was found at the 34% (w/w) sucrose layer (1.141 grams per cubic centimeter) which co-fractionated with the pH 6.5-ATPase.

Identification of plasma membrane was performed by the combination of phosphotungstic acid-chromic acid stain and specific binding of N-1-naphthylphthalamic acid. Based on morphometrical observations after phosphotungstic acid-chromic acid stain, the isolated plasma membrane consisted mostly of vesicles of high purity. The isolated plasma membrane also showed extremely high specificity for N-1-naphthylphthalamic acidbinding, 10-fold higher than other membranes. It was also confirmed that there is a distinct difference in properties between plasma membrane and other membranes. The endomembranes such as from chloroplasts, mitochondria, and endoplasmic reticulum were observed to be highly sensitive to Zn²⁺ ion and lower pH, which resulted in an abrupt aggregation of membranes. On the contrary, plasma membrane was very stable to these treatments and no aggregation was observed. These unique properties of isolated plasma membrane are generally observed in a wide variety of plant species and can be utilized for the assessment of the purity of preparations of isolated plasma membranes and for their identification.

     

Opaline Silica Deposition in Rye (Secale cereale L.)

The types of opaline silica-bodies (opal phytoliths) which occurin the mature prophylls, radical and culm leaves, culms, andinflorescence bracts of rye (Secale cereale L.) are describedand figured. Silica-bodies are absent from the coleoptile, andthe adaxial epidermis of the prophylls, leaf sheaths, and inflorescencebracts. The stages of silica-body formation in young radicalleaf sheaths are also described. Alternative hypotheses forthe origin of silica-bodies are discussed.     

Distribution and function of actin in the developing stomatal complex of winter rye (Secale cereale cv. Puma)

Summary The dynamics of actin distribution during stomatal complex formation in leaves of winter rye was examined by means of immunofluorescence microscopy of epidermal sheets. This method results in actin localization patterns that are the same as those seen with rhodamine-phalloidin staining, but are more stable. During stomatal development MFs are extensively rearranged, and most of the time the orientation or placement of MFs is distinctly different from that of MTs, the exception being co-localization of MTs and MFs in phragmoplasts. Although MFs show an orientation similar to that of MTs in interphase guard mother cells, no banding of MFs into anything resembling the interphase MT band is observed. From prophase to telophase, a distinct, dense concentration of MFs is found in subsidiary cell mother cells (SMCs) between the nucleus and the region of the cell cortex facing the guard mother cell. Cytochalasin B treatment causes incorrect positioning of the SMC nucleus/daughter nuclei and abarrent placement and orientation of the new cell wall that forms the boundary of the subsidiary cell at cytokinesis. These results suggest that MFs are involved in maintaining the SMC nucleus in its correct position and the SMC spindle in the correct orientation relative to the division site previously delineated by the preprophase band. Because these MFs thus appear to assure that the SMC phragmoplast begins to form in the correct orientation near the division site to which it needs to grow, we suggest that MFs are involved in control of correct placement and orientation of the new cell wall of the subsidiary cell.Abbreviations CB cytochalasin B - DIC differential interference contrast - DMSO dimethylsulfoxide - MBS m-maleimidobenzoyl-N-hydroxylsuccinimide ester - MF microfilament - MT microtubule - PBS phosphate buffered saline - SMC subsidiary cell mother cell Dedicated to the memory of Professor Oswald Kiermayer     

Comparisons of Plasma Membranes from Shoots and Roots of Winter Rye (Secale cereale L. cv. Puma): Polypeptide Composition, ATPase Activity and Specific Naphthylphthalamic Acid Binding Capacity

Polypeptide compositions, ATPase characteristics, and the N-1-naphthylphthalamicacid binding capacity of plasma membranes prepared from winterrye (Secale cereale L. cv. Puma) shoots and roots were examinedand compared. Some unique polypeptides were revealed in each plasma membraneby one- and two-dimensional slab gel electrophoresis. A differencewas also detected in glycopeptide compositions. The plasma membranesfrom both organs contained Mg²⁺-stimulated ATPase exhibitingslightly different properties in the divalent cation specificityand the kinetic constants. The ATPase activities from both organsshowed a similar optimum pH around 6.5, simple Michaelis-Mentensaturation with increasing ATP-Mg concentrations, and littleK⁺-stimulation at the optimum pH. Both ATPases were inhibitedby orthovanadate, however, the degree of inhibition was a littledifferent in each membrane sample. The specific N-1-naphthylphthalamicacid binding capacity in the shoot plasma membrane was 2.6-foldhigher than that in the root plasma membrane. These results suggest that polypeptide compositions of plasmamembranes vary corresponding with a difference in the physiologicalfunctions of plasma membranes between shoots and roots of winterrye. ¹ Contribution No. 2670 from the Institute of Low TemperatureScience. (Received May 17, 1984; Accepted October 9, 1984)     

Heat Tolerance of Cold Acclimated Puma Winter Rye Seedlings and the Effect of a Heat Shock on Freezing Tolerance

An increase in tolerance to one form of abiotic stress oftenresults in an increase in tolerance to another stress. The heattolerance of Puma rye (Secale cereale) was determined for seedlingseither not cold hardened or hardened under either controlledenvironmental or natural conditions. The heat tolerance wasdetermined either as a function of time at 42°C or the abilityto tolerate a maximum temperature. The seedlings were eithernot heat preconditioned or heat preconditioned before the heatstress. In all cases cold hardened seedlings were more heattolerant than non or partially cold hardened seedlings. Heatpreconditioning had no effect on the heat tolerance of naturallycold hardened seedlings. In contrast, seedlings cold hardenedin a controlled environment chamber, then heat preconditioned,were more heat tolerant than non preconditioned seedlings. Aheat shock of 36°C for 2 h increased the freezing toleranceof non hardened seedlings from –2.5°C to –4.5°C.Analysis of heat shock protein 70 (HSP70) gene expression indicatedthat the HSP70 gene was not induced by cold acclimation andtherefore not directly involved in the increased thermo toleranceobserved. A number of heat stable proteins, simple sugars andlong chain carbohydrate polymers accumulated during the coldacclimation process and may have a role in increasing heat toleranceas well as freezing tolerance. These data suggest cold hardeningincreases heat tolerance, however, a heat shock to non acclimatedseedlings only marginally increased the freezing tolerance ofPuma rye seedlings. ³Present address: Agriculture and Agri-Food Canada, 107 SciencePlace, Saskatoon SK S7N 0X2, Canada.     

Freeze-Induced Membrane Ultrastructural Alterations in Rye (Secale cereale) Leaves

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.     

Relationship Between the Development and Growth of Rye (Secale cereale L.) and the Potassium Concentration in Solution

The development and growth of rye (Secale cereale L. cv. Rheidol)was studied in seedlings grown hydroponically in complete nutrientsolutions containing between 10 and 600 µM K⁺. The phyllochron(defined as the interval between the appearance of successiveleaves) was used as a developmental timescale to compare plants.The pattern of both shoot and root development was strictlyordered on a phyllochron basis and was unaffected by solutionK⁺ concentration, with the exception that tillers in plantsgrown at the lowest K⁺ concentrations were occasionally eithernot initiated or aborted at an early stage of development. However,both the rate of leaf appearance on the main stem and successivetillers and the rate of tiller appearance were slower in plantsgrown at lower K⁺ concentrations. The rate of leaf appearanceon the main stem was reduced to below 90% of its maximal valueat solution concentrations below about 50 µM K⁺. Plantrelative growth rate (RGR) was also reduced by lowering theK⁺ concentration of the nutrient solution and fell to below90% of its maximal value at solution concentrations below about200 µM K⁺. There was a complex relationship between tissueK⁺ concentration and the K⁺ concentration of the nutrient solution,which differed between leaves and root. Leaf K⁺ concentrationincreased steadily from about 50 µmol g^-1 f. wt to about200 µmol g^-1 f. wt as solution K⁺ concentration was increasedfrom 10 to 400 µM. In contrast, root K⁺ concentrationexhibited a sigmoidal dependence on solution K⁺ concentration,maintaining a minimal value of approximately 20 µmol g^-1f. wt at concentration below 100 µM K⁺, then increasingprogressively to about 120 µmol g^-1 f. wt at a solutionconcentration of 600 µM K⁺. The 'critical' leaf K⁺ concentration,i.e. the concentration at which either plant RGR or plant developmentwas reduced 90% of its maximal value, was 86 µmol g^-1f. wt for plant RGR and 150 µmol g^-1 f. wt for plant development.The 'critical' root K⁺ concentration was 24 µmol g^-1 f.wt K⁺ for both RGR and development. A decline in tissue K⁺ concentrationbelow these thresholds reduced plant growth considerably. RootK⁺ concentration was a sensitive indicator of the K⁺ statusof the plant with respect to potential growth since plant growthdeclined abruptly as root K⁺ concentration approached its 'critical'value, whereas plant growth showed a less defined relationshipwith shoot K⁺ concentration.Copyright 1993, 1999 Academic Press Critical K⁺ concentration, development, potassium, relative growth rate (RGR), rye, Secale cereale L. cv. Rheidol     

Microdissection and Recovery of 1R Chromosome in Rye ( Secale cereale )

The technique of mieromanipulaion has been used to establish a system of single chromosome mierodissection. According to the standard karyotype of rye ( Secale cereale L. ), 1R chromosome carrying disease-resistant gene has been identified, microdissected and transferred into Ep-pendorf tube using mieromanipulator. The results showed that IR chromosome in cells pretreated with α-bromonaphthalene could be identified quickly and the efficiency of microdissection was greatly improved when the technique of wall degradation and hypotonic treatment was applied.     

The Pattern and Sequence of Opaline Silica Deposition in Rye (Secale cereale L.)

The pattern of opaline silica deposition in the leaves and internodesof rye (Secale cereale L.) has been studied by means of countsof silica-bodies in cleared epidermal preparations. Silica depositionoccurs during the maturation process when the leaves are fullyexpanded. The increase in total silica content and changes inthe ‘free’ and residual silica fractions of theleaves during their growth period have been determined usinga colorimetric estimation.     

Mechanical Transmission of Barley Mild Mosaic Virus (BaMMV) to Rye (Secale cereale L.)

After mechanical spraygun inoculation barley mild mosaic virus (BaMMV) was detected in barley cv. ‘Gerbel’ (control) as well as in rye cv. ‘Somro’, but not in wheat cv. ‘Kanzler’ and oat cv. ‘Alfred’. ELISA values of infected barley and rye were similar. Furthermore, infected rye plants developed symptoms typical for barley yellow mosaic virus infection.     

Extraction and Isolation of Antifreeze Proteins from Winter Rye (Secale cereale L.) Leaves

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.     

Replicon size and mean rate of DNA synthesis in rye (Secale cereale L. cv. Petkus Spring)

Chromosomal DNA replication was investigated in root meristem cells of Secale cereale L. cv. Petkus Spring using DNA fibre auto-radiography. At 23 ° the mean rate of replication, per single replicon fork, was 12.1 m/h. Replicon size was between 20–25 m. These results are compared with corresponding measurements for other angiosperm species.     

Site of Synthesis of NADPH: Protochlorophyllide Oxidoreductase in Rye (Secale cereale)

The site of synthesis of the plastid membrane-located enzyme, protochlorophyllide reductase, has been determined. Plastid ribosome-deficient and normal rye (Secale cereale L., cv Rheidol) plants were grown in darkness at 33°C and 22°C, respectively. Extracts from these plants were analyzed for the levels of different ribosomal RNAs and cytochrome f and the activity of a number of enzymes with well-established sites of synthesis. The results confirmed that the higher temperature had induced a specific inhibition of protein synthesis in the plastids. The activity and level of protochlorophyllide reductase was unaffected by growth at the higher temperature, suggesting it to be a cytoplasmically synthesized enzyme.     

Characterization and Quantification of Intrinsic Ice Nucleators in Winter Rye (Secale cereale) Leaves

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.     

The Relation between Cytomixis and Variation of Chromosome Numbers in Pollen Mother ceils of Rye (Secale cereale L.)

Cytomixis is a spontaneous and normal process occurring through the formation of plasma channels between neighbouring pollen mother cells (PMCs). This phenomenon was observed in the PMCs of 2 variaties of Secale cereale which had been collected from Gausu and Shanxi Provinces. The process had led to the formation of up to 6.37% of PMCs with chromosome numbers deviating from the normal haploid number n=7. The abnormal PMCs contained chromosome numbers ranging from 5–9 in metaphase Ⅰ. Abnormal PMCs with decreased and increased chromosome numbers could be the result of cytomixis. The cytological consequences and the evolutionary significance of cytomixis are discussed.     

The Banding Pattern of the Rye (Secale cereale) Chromosomes and Its Application

The Giemsa C-banding technique has been used in this paper for analysis of chromosome banding pattern, and the changes of the chromosome structures of irradiated rye and wheat-rye were identified preliminarily. Heterochromatin polymorphism of rye was also discussed.