Ultrastructural changes arising from freezing of leaf blade cells of rye (Secale cereale): an investigation using freeze-substitution

The survival at sub-zero temperatures of leaf blade cells of rye ( Secale cereale L. cv. Voima), which had not been cold acclimated, was determined by measuring the efflux of ninhydrin-positive substances: 50% of the cells were dead at −4°C (LT₅₀) and none survived at −12°C or below. Examination of ultrastructural changes during cold hardening and freezing injury requires frozen tissues prepared for transmission electron microscopy without thawing. Specimens were prepared from leaf blade segments at room temperature, −4°C or −12°C by plunge freezing at 3 m s⁻¹ into a cooling medium at −170°C followed by freeze-substitution in acetone with OsO₄ fixation. Comparisons of room temperature specimens were made with those prepared by chemical fixation using glutaraldehyde/paraformaldehyde/tannic acid. On freezing to −12°C, the cells were severely dehydrated and distorted, the vacuoles severely shrunken and the cytoplasm and mitochondria disorganized whereas the chloroplasts were little affected. On freezing to −4°C, some cells were as disorganized as those at −12°C, others were relatively intact, and some showed evidence of intracellular ice crystal formation.     

Cold-induced changes in the polysome pattern and protein synthesis in winter rye (Secale cereale) leaves

Cold-induced changes in the polysome pattern and protein synthesis were analyzed in winter rye, Secale cereale L. cv. Voima, during one week's cold stress treatment, which was performed by transferring the 7-day-old plants from the greenhouse (25°C, long-day conditions) to 3°C and a photoperiod of 10. 5 h. Freezing resistance determined by electrolyte leakage increased significantly upon cold stress starting from LT₅₀ value –5°C. and reaching –9°C on the day 7 of cold exposure. After 4 weeks at low temperature, plants reached an LT₅₀ of –12°C. The polysome content increased markedly during cold stress compared to the control plants. After 2 weeks of cold treatment the polysome content decreased to the same level as that in control plants. The size-class distribution of polysomes showed a high proportion of large protein synthesizing polysomes in cold-stressed plants. After 2 weeks the values were comparable to those in control plants. Cold-induced proteins were detected using ³⁵S-labelled methionine for in vitro translations. At least 2 new polypeptides, M_r 30000 and 18000, were induced on the first day of cold stress and continued to be expressed at low temperatures 4 weeks later.     

Gibberellin status and responsiveness in shoots of tall and dwarf genotypes of diploid rye (Secale cereale)

The recessive dwarfing alleles of rye ( Secale cereale L.), ct1 and ct2 , caused a 35–55% reduction in the length of leaf 2 compared with corresponding tall lines grown at both 10°C and 20°C. The dwarf lines were 45–50% as responsive to applied GA₃ as the tall lines at 20°C but the absolute GA-responsiveness of the dwarfs was greater at 10°C than at 20°C. There was no significant difference in the contents of GA₁₉, GA₂₀, GA₂₉, GA₁, GA₃ and GA₈ in the leaf extension zone of tall and dwarf seedlings grown at 20°C. It was concluded that the mechanism whereby GA homeostasis is maintained is functional in both tall and dwarf lines despite marked differences in leaf extension rate. The recessive rye mutations may cause loss of function late in the GA-cell elongation pathway or, alternatively, indirectly affect GA-responsiveness in vegetative tissues. The genetic and physiological evidence indicates that ct1 and ct2 are unrelated to the GA-insensitive Rht genes in hexaploid bread wheat.     

Linkage relationships of esterase loci in rye (Secale cereale L.)

Summary Genetic analysis of esterase polymorphism in rye inbred lines with isoelectric focusing in polyacrylamide flat gels yielded evidence for the existence of at least ten esterase loci, Est 1–Est 10. The loci can be attributed to four different linkage groups (Est 1/ Est 2/Est 3/Est 5/Est 6/Est 7), (Est 4), (Est 8/Est 9), and (Est 10). Loci Est 5/Est 6/Est 7 and Est 8/Est 9, respectively, are tightly linked with a maximum recombination frequency of 0.2% and can therefore be regarded as compound loci which possibly originated in tandem duplications.     

Interactive effects of Al, Ca and other cations on root elongation of rice cultivars under low pH

BACKGROUND AND AIMS: As with other crop species, Al tolerance in rice (Oryza sativa) is widely different among cultivars, and the mechanism for tolerance is unknown. The Ca2+-displacement hypothesis, that is, Al displaces Ca2+ from critical sites in the root apoplast, was predicted to be the essential mechanism for causing Al toxicity in rice cultivars. If displacement of Ca is an essential cause of Al toxicity in rice, Al toxicity may show the same trend as toxicities of elements such as Sr and Ba that are effective in displacing Ca. METHODS: The interactive effects of Al, Ca, Sr and Ba on root elongation of rice cultivars with different Al tolerances were evaluated in hydroponic culture. Al and Ca accumulation in root tips was also investigated. KEY RESULTS AND CONCLUSIONS: Not only Al but also Sr and Ba applications inhibited root growth of rice cultivars under low Ca conditions. As expected, rice cultivars more tolerant of Sr and Ba were also tolerant of Al (japonica > indica). Although Mg application did not affect Sr or Ba toxicity, Mg alleviated Al toxicity to the same level as Ca application. In addition, Ca application decreased the Al content in root tips without displacement. These results suggest that Ca does not have a specific, irreplaceable role in Al toxicity, unlike Sr and Ba toxicities. Alleviation of Al toxicity with increasing concentrations of Ca in rice cultivars is due to increased ionic strength, not due to decreased Al activity. The difference in Al tolerance between indica and japonica cultivars disappears under high ionic strength conditions, suggesting that different electrochemical characteristics of root-tip cells are related to the significant difference in Al tolerance under low ionic strength conditions.     

Effect of cold exposure on cortical microtubules of rye (Secale cereale) as observed by immunocytochemistry

The responses of cortical microtubules to sub-zero temperatures were examined in non-acclimated (NA) and cold-acclimated (CA) rye ( Secale cereale L. cv. Voima) leaf and root cells, and in protoplasts isolated enzymatically from leaves. Responses of leaf and root cells to hypertonic solutions equivalent to the dehydration response of freezing (P. L. Steponkus and D. V. Lynch 1989. J. Bioenerg. Biomembr. 21: 21–41) were also examined. At the respective growth temperatures both NA and CA leaf and root cells had typical organization and abundance of cortical microtubules as observed by indirect immunofluorescence (IIF) staining. Unchanged microtubule arrays were still present in CA leaf cells after -4°C treatment, while in leaf cells of NA plants and in the root cells of both NA and CA plants microtubules were shorter and less abundant. After -10°C treatment the cortical microtubules were almost totally depolymerized in both types of root cells and in leaf cells of NA plants, while CA leaf cells still had abundant cortical microtubule arrays. Semiquantitative analyses of cortical microtubules (MTs) of protoplasts confirmed the findings with intact leaf cells. Hypertonic treatment of NA and CA leaf cells gave similar effects as exposure of cells to sub-zero temperatures. However, after the hypertonic treatment, more microtubules remained present in the CA root cells than in the NA root cells, suggesting that also in root cells cold acclimation increases the dehydration stability of MTs. In conclusion, cold acclimation induces both greater frost stability and greater osmotic tolerance in the cortical microtubules of the leaf cells, and greater osmotic tolerance in the microtubules of the root cells in winter rye.     

B-chromosomes in inbred lines of rye (Secale cereale L.)

B-chromosomes from an experimental population of the Japanese JNK strain of rye, isogenic for its Bs, have been backcrossed into twelve different inbred lines. The experiment provides a way to study the effects of the Bs against a range of homozygous A-chromosome backgrounds. This publication deals with vigour and fertility: it shows that the rye Bs fit a parasitic model, and that they interact in their effects with the A-chromosome background genotype.     

Phenolic substances as allelopathic agents arising during the degradation of rye (Secale cereale) tissues

Rye seedlings, tillering plants and crop residues were allowed to decompose in model incubation experiments. Young tissues gave rise to high concentrations of allelochemicals, whereas crop residues did not produce inhibitors. Seven phenolic acids were identified in the investigated materials; p-hydroxybenzoic protocatechuic, gallic, vanillic, syringic, p-coumaric, ferulic as well as benzoic acid. However, neither the level of these acids nor the total content of phenolic compounds corresponded to the level of phytotoxicity determined in bioassays. This demonstrated that, apart from phenolics, other unidentified water-soluble organic compounds were also responsible for the toxicity of rye decomposition products. The study was conducted within program CPBP 04.10.03. The study was conducted within program CPBP 04.10.03.     

B-chromosomes in inbred lines of rye (Secale cereale L.)

B chromosomes from an experimental population of the Japanese JNK strain of rye, isogenic for its Bs, have been backcrossed into twelve different inbred lines. The experiment is a way of studying the effects of the Bs against a range of different homozygous A chromosome backgrounds. This publication deals with pairing effects of both the As and the Bs, and their interactions, and with pollen mitosis. At meiosis there is a genotypic component to B effects, and they do not appear to act solely through a physical disturbance within the nucleus. In pollen the Bs are always present in more than 50% of the grains regardless of their pairing behaviour during meiosis; this result fits with a parasitic model of the activity of rye Bs.     

Mapping of three self-fertility mutations in rye (Secale cereale L.) using RFLP, isozyme and morphological markers

 Three mutations determining self-fertility at the S, Z and S5 self-incompatibility loci on chromosomes 1R, 2R and 5R of rye, respectively, were mapped using three different F₂ populations. There was a close linkage of one isozyme and four RFLP markers, and no recombinant plants were detected. These markers are Prx7, Xiag249 and Xpsr634 for the S locus (1R), Xbcd266 for the Z locus (2R) and Xpsr100 for the S5 locus (5R). Linkage data for markers associated to the self-fertility mutations at the S, Z and S5 loci were calculated and compared with genetic maps computed by MAPMAKER multipoint analysis. Received: 8 October 1997 / Acepted: 26 November 1997     

Phosphorylation of pollen proteins in relation to self-incompatibility in rye (Secale cereale L.)

The gametophytic two-locus self-incompatibility (SI) system in rye was investigated in view of a possible involvement of protein phosphorylation and Ca²⁺ as constituents of a signal transduction mechanism. Phosphorylation kinetics in pollen grains was found to be significantly different after in vitro treatment of pollen with either cross or self stigma proteins, with a pronounced phosphorylation activity in self-treated pollen grains. Loss of SI in self-compatible (SC) mutants was associated with a significantly decreased basic phosphorylation activity in untreated pollen grains as compared to SI genotypes. Separation of phosphorylated pollen proteins by SDS-PAGE reveals four major proteins in the MW range of 43–82 kDa which were differently phosphorylated in SI vs SC genotypes as well as in cross vs self-treated pollen grains. Application of different protein kinase inhibitors and the Ca²⁺ antagonists verapamil and La³⁺ to isolated stigmas resulted in an inhibition of the SI response in in vitro self-pollination. The role of protein kinases and Ca²⁺ as constituents of a putative SI-specific signal transduction mechanism is discussed.     

Acclimation of potassium influx in rye (Secale cereale) to low root temperatures

The influx of K⁺(⁸⁶Rb⁺) into intact roots of rye (Secale cereale L. cv. Rheidal) exposed to a differential temperature (DT) between the root (8° C) and shoot (20° C) is initially reduced compared with warm-grown (WG) controls with both shoot and root maintained at 20° C. Over a period of 3 d, however, K⁺-influx rates into DT plants are restored to levels similar to or greater than those of the WG controls, the absolute rates of K⁺ influx being strongly dependent upon the shoot/root ratio. Acclimation in DT plants results in a reduction of K⁺ influx into the apical (0–2 cm) region of the seminal root which is associated with a compensatory increase in K⁺ influx into the more mature, basal regions of the root. Values of V _max and apparent K _m for K⁺ influx into DT plants were similar to those for WG plants at assay temperatures of 8° C and 20° C except for an increase in the apparent K _m at 8° C. The influx of K⁺ from solutions containing 0.6 mol·m^-3 K⁺ into both WG and DT plants was found to be linearly related to assay temperature over the range 2–27° C, and the temperature sensitivity of K⁺ influx to be dependent upon shoot/root ratio. At high shoot/root ratios, the ratio of K⁺ influx at 20° C:K⁺ influx at 8° C for WG plants approached a minimum value of 1.9 whereas that for DT plants approached unity indicating that K⁺ influx into DT plants has a large temperature-insensitive component. Additionally, when plants were grown in solutions of low potassium concentration, K⁺ influx into DT plants was consistently greater than that into WG plants, in spite of having a greater root potassium concentration ([K⁺]int). This result indicates some change in the regulation of K⁺ influx by [K⁺]int in plants exposed to low root temperatures. We suggest that K⁺ influx into rye seedlings exposed to low root temperatures is regulated by the increased demand placed on the root system by a proportionally larger shoot and that the acclimation of K⁺ influx to low temperatures may be the result of an increased hydraulic conductivity of the root system.Abbreviations DT differential temperature pretreatment - [K⁺]int root potassium concentration - [K⁺]ext potassium concentration of nutrient medium - WG warm-grown pretreatment     

Genetics and molecular mapping of a male fertility restoration locus (Rfg1) in rye (Secale cereale L.)

 A gene determining the restoration of cytoplasmic genic male sterility (CMS) caused by the Gülzow (G)-type cytoplasm was mapped by analyzing an F₂ and F₃ population comprising 140 and 133 individual plants, respectively. The target gene, designated Rfg1, was mapped on chromosome 4RL distally to three RFLP (Xpsr119, Xpsr167, Xpsr899) and four RAPD (XP01, XAP05, XR11, XS10) loci. Xpsr167 and Xpsr899 are known to be located on the segment of chromosome 4RL which was ancestrally translocated and is homoeologous to the distal end of other Triticeae 6S chromosomes. It is suggested that Rfg1 may be allelic to the gene determining the restoration of rye CMS caused by the Pampa (P) cytoplasm (chromosome 4RL) and to Rfc4 that on rye addition lines of chromosome 4RL restores male fertility of hexaploid wheat with T. timopheevi cytoplasm. Homoeoallelism to two loci for cytoplasmic-male-sterility restoration on chromosomes 6AS and 6BS in hexaploid wheat is also suggested. Received: 1 December 1997 / Accepted: 10 February 1998     

Ca2+-binding sites and Ca2+-ATPase activity in barley root tip cells

Summary Different cytochemical methods were employed to demonstrate the existence of Ca²⁺-binding sites (Ca²⁺-bs) at the membranes of barley root tip cells, involving addition of CaCl₂ (10 mM or 1 mM) to all aqueous solutions used for tissue processing for electron microscopy, treatment of ultrathin sections by Ca-chelating agents, enzymic digestion of ultrathin sections and modification of Wachstein-Meisel procedure for localization of Ca²⁺-dependent ATPase activity. Addition of 10 mM CaCl₂ to the fixatives and rinsing solutions causes electron-dense globules (EDG) to be formed in a variety of cells, those in cortical cells being associated mainly with the plasma membranes, in root cap cells with the plasmalemma as well as with majority of intracellular membranes. The obligatory presence of EDG at the membranes of Golgi vesicles and secretory vesicles approaching plasmalemma was revealed in the secreting root cap cells. Besides, electron opaque connecting material was found between the plasmalemma and adjacent secretory vesicle membranes. In true meristematic cells Ca-supplemented solutions induce formation of EDG localized at the ER membranes, and nuclear and plastid envelopes. In root cells of seeds germinated in the presence of 1 mM CaCl₂ electron opaque deposits were found only in local areas of plasmalemma collars around plasmodesmata neck regions, contacting the terminals of subsurface ER channels. In control speciemens (germination, fixation and washing without added CaCl₂) EDG were absent in cortical and ground meristem cells, but present in root cap cells, although their number and average size were greatly reduced.Treatment of thin sections by 10mM EGTA or EDTA led to complete removing of EDGs, electron-transparent holes replacing them. Digestion by a variety of proteolytic enzymes and by phospholipase A induced partial destruction of EDG matrices, confirming the presence of protein as well as of phospholipid membrane components. Visualization of electron-dense granular product of cytochemical Ca-ATPase reaction at the same membrane areas where EDG were located suggests that one of the Ca-binding proteins in EDG may represent Ca-ATPase.It is proposed that EDG at plant cell membranes have a certain resemblance to the Ca²⁺-bs revealed by the same method on plasma membrane of a variety of animal cells. The data obtained are discussed regarding possible regulatory roles of calcium ions in plant cells, especially in exocytotic secretion.     

Nitric oxide signalling in plants: interplays with Ca2+ and protein kinases

Much attention has been paid to nitric oxide (NO) research since its discovery as a physiological mediator of plant defence responses. In recent years, newer roles have been attributed to NO, ranging from root development to stomatal closure. The molecular mechanisms underlying NO action in plants are just begun to emerge. The currently available data illustrate that NO can directly influence the activity of target proteins through nitrosylation and has the capacity to act as a Ca2+-mobilizing intracellular messenger. The interplay between NO and Ca2+ has important functional implications, expanding and enriching the possibilities for modulating transduction processes. Furthermore, protein kinases regulated through NO-dependent mechanisms are being discovered, offering fresh perspective on processes such as stress tolerance.     

A consensus linkage map of rye (Secale cereale L.) including 374 RFLPs, 24 isozymes and 15 gene loci

 Consensus linkage maps were constructed for all seven rye chromosomes using 12 basic RFLP maps. The maps presented contain a total of 413 markers. The number of markers per chromosome varies from 41 (chromosome 3R) to 83 (chromosome 1R). In addition to 374 RFLP and 24 isozyme markers 15 gene loci were incorporated, determining the traits reduced plant height, self fertility, male sterility restoration, vernalization response, resistance against powdery mildew, chlorophyll deficiency, hairy leaf sheath, hairy peduncle, waxy endosperm, waxless plant and absence of ligules. The maps presented allow the selection of markers for the fine mapping of certain regions of the rye genome. In terms of the known chromosomal rearrangements within the Triticeae its utilization can also be extended for mapping in wheat and barley. Received: 13 February 1998 / Accepted: 26 May 1998     

A new type of cytoplasmic male sterility in rye (Secale cereale L.): analysis of mitochondrial DNA

Summary Mitochondrial (mt) DNA of a new type of rye cytoplasm (Gülzow, G) that induces cytoplasmic male sterility (CMS) was analyzed and compared with rye mtDNAs of different origins MtDNA of the G type was easily distinguishable from mtDNA of another CMS source, Pampa (P) type, and from mtDNA of fertile lines with respect to restriction fragment patterns and hybridization with mitochondrial genes. The results of the molecular analyses indicate a close, but not identical relationship between the mtDNA of the G type cytoplasm and that of cv Pluto.     

Chromosomal location of esterase,peroxidase and phosphoglucomutase isozyme structural genes in cultivated rye (Secale cereale L.)

Summary Isoelectric focusing of esterase (EST), peroxidase (PRX), and phosphoglucomutase (PGM) isozymes in Chinese Spring wheat, Imperial rye and several Chinese Spring/Imperial and Holdfast/King II addition, translocation and substitution lines revealed the chromosomal location of nine Est loci previously described and of one Prx and Pgm locus. Loci Est1, Est2, Est3, Est5, Est6 and Est7 were found on chromosome arm 5RL, Est8 and Est9 on chromosome 6R in Imperial rye, and the Est10 locus on chromosome arm 4RL in Imperial rye and King II rye. A discrepancy was found between the chromosomal location of the Prx locus in Imperial where chromosome 2R was responsible for the expression of the peroxidase enzyme, and King II with chromosome 1R carrying the Prx gene. As a possible explanation, the occurrence of translocation events during the production of wheat/rye aneuploid lines is discussed. The rye Pgm locus could be associated with chromosome 4RS in Imperial and King II rye. Except for the location of Est loci on chromosome 5RL, the results reported in this paper lend further evidence for the assumed homoeology relationships between the chromosomes of Triticinae and for the conservation of gene synteny groups during the evolution of the Triticeae tribe.     

Genetic factors influencing regeneration ability in rye (Secale cereale L.). I. Immature inflorescences

Immature inflorescences of ten rye inbred lines (inbred degree S10 and S11) were cultured on solidified MS medium supplemented with 3.0 mg/dm³ of 2,4-D. According to their capability for callus production explants were classified into two groups : responsive (giving weak or intensive callus production) and non responsive (lack of callus formation). After transferring responsive material into hormone-free medium the regeneration of roots or shoots from the intensive growing callus was observed. Consistent differences between lines in the portion of explants with a certain response were found. They were divided into five groups reacting in the same way. Lines with different in-vitro response were crossed in an incomplete diallel. F₁, F₂ and F₃ generations were analyzed and the following conclusions drawn: the ability for plant regeneration from immature inflorescences in rye is determined by numerous loci, has a recessive character, and both callus production and regeneration suppression may be controlled by complementary genes.     

A genetic map of rye (Secale cereale L.) combining RFLP, isozyme, protein, microsatellite and gene loci

Among the cereals, rye (Secale cereale L.) can be grown under extreme climatic and poor soil conditions and, is a major crop in North Europe. In the present paper, we report the development of a genetic linkage map of rye using a pooled F₂ mapping population created from a reciprocal cross of two self-fertile inbred lines. The 183 mapped markers consist 139 RFLPs, 19 isozyme and protein markers, 13 microsatellites, 10 known function sequences and two morphological genes. The markers are randomly distributed on the seven chromosomes with a maximum of 38 on chromosome 5R and a minimum of 19 on chromosome 3R. In addition, 23 gene loci and 25 quantitative trait loci were aligned to chromosome regions. For some of the mapped or aligned genes comparable loci are present in other cereals. The homoeologous relationships of these loci are discussed. The potential of the new map for further genetic studies is outlined. Received: 11 May 2000 / Accepted: 12 July 2000