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.     

Aluminium toxicity in rye (Secale cereale): root growth and dynamics of cytoplasmic Ca2+ in intact root tips

Aluminium (Al) toxicity in rye (Secale cereale L.), an Al-resistant crop, was examined by measuring root elongation and cytoplasmic free activity of calcium ([Ca2+]cyt) in intact root apical cells. Measurement of [Ca2+]cyt, was achieved by loading a Ca2+-sensitive fluorescent probe. Fluo-3/AM ester, into root apical cells followed by detection of intracellular fluorescence using a confocal laser scanning microscope. After 20 min of exposure to 50 microM Al (pH 4-2) a slight increase in [Ca2+]cyt of root apical cells was observed, while the response of [Ca2+]cyt to 100 microM Al (pH 4.2) was faster and larger ([Ca2+]cyt increased by 46% in 10 min). Increases in [Ca2+]cyt were correlated with inhibition of root growth, generally measurable after 2 h. Addition of 400 microM malic acid (pH 4.2) largely ameliorated the effect of 100 microM Al on [Ca2+]cyt in root apical cells and protected root growth from Al toxicity. These results suggest that an increase in [Ca2+]cyt in root apical cells in rye is an early effect of Al toxicity and is followed by the secondary effect on root elongation.     

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.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

Immunolabeled microtubules and microfilaments are visible in multiple cell layers of rye root tip sections

Summary A protocol was developed to observe plant microtubules and actin microfilaments in large tissue samples without physical sectioning. Rye (Secale cereale L. cv. Rymin) root tip pieces from two-day-old seedlings were fixed and processed for immunolabeling. Incubation times of 24–48 h were required to insure adequate penetration of fixatives, antibodies, and washing buffers. Clearing of the tissue with methyl salicylate reduced background auto-fluorescence that would otherwise interfere with the resolution of cytoskeletal structures. Microtubules or microfilaments in 5–7 cell layers were visualized using the optical-sectioning capability of laser scanning confocal microscopy (LSCM) and projected as three-dimensional images. The three-dimensional character of the cytoskeletal elements is retained when viewing stained cells of intact tissue. The net-like character of a microfilament array radiating out from a single point into the cytoplasm is maintained when the cells are stained in intact root tip pieces and imaging is accomplished in situ.Abbreviations Cy3 cyanine 3.18-conjugated goat anti-mouse IgG - FITC-M fluorescein isothiocyanate conjugated anti-mouse IgG - IFB immunofluorescence buffer - LSCM laser scanning confoeal microscopy - TPBS phosphate-buffered saline with 0.1% Triton X-100     

Differential expression of two β-amylase genes of rye during seed development

The expression of two β-amylase loci was analysed in the developing seeds of two inbred lines of rye (Secale cereale L.), one of which was a β-amylase deficient mutant. Enzymatic activity and the contents of enzymatic protein and mRNA specific for each of an endosperm-characteristic and ubiquitous β-amylase were determined throughout the course of caryopsis development. Both loci were expressed in the developing normal line caryopses according to different temporal and quantitative patterns. The ubiquitous enzyme-specific locus β-Amy 2 was expressed earlier; both mRNA and enzymatic protein accumulated to a maximum extent at 10 to 15 days after pollination. In contrast, the highest content of mRNA for endosperm β-amylase (encoded by the β-Amy I locus) was found 20 days after pollination, and the corresponding enzymatic protein accumulated throughout seed development. The expression of the β-Amy I locus was 30- to 40-fold higher than that of the β-Amy 2 locus in terms of maximum specific mRNA accumulation. The expression product of only the β-Amy 2 locus was found in the developing mutant line caryopses. The expression pattern of this locus was similar in the developing normal and mutant line seeds in terms of the temporal accumulation of mRNA and enzymatic protein. However, an approximately 4-fold higher level of ubiquitous β-amylase-specific mRNA was found in the mutant than in the normal line caryopses, and the content of ubiquitous β-amylase protein decreased to near zero at seed maturity in the mutant line, but not in the normal line, caryopses. The enzymatic activities of both β-amylases appeared to be regulated at the level of accumulated enzymatic protein.     

Photoinhibition of isolated mesophyll cells from cold-hardened and non-hardened winter rye

Cold-hardened rye leaves have been shown to be more resistant to low temperature photoinhibition than non-hardened rye leaves. Isolated mesophyll cells from winter rye (Secale cereale L. cv. Musketeer) were exposed to photoinhibitory light conditions to estimate the importance of leaf morphology and leaf optical properties in the resistance of cold-hardened rye leaves to photoinhibition. Cold-hardened rye cells showed more resistance to photoinhibition than non-hardened rye cells when monitored with chlorophyll a variable to maximal fluorescence ratio (F_v/F_m). Thus, leaf morphology does not contribute to the resistance of cold-hardened rye leaves to low temperature photoinhibition. However, cold-hardened and non-hardened rye cells showed a similar extent of photoinhibition when photsynthetic CO₂ fixation rates were measured. They also showed the same capacity to recover from photoinhibition. During both photoinhibition and recovery, F_v/F_m and light limited CO₂ fixation rates showed different kinetics. We propose that inactivation and subsequent reactivation during recovery of some light activated Calvin cycle enzymes explain the greater extent of photoinhibition of light limited CO₂ fixation and its faster recovery compared to F_v/F_m kinetics during photoinhibition.     

X-ray microanalytical (EDX) investigation of potassium distributions in mesophyll cells of non-acclimated and cold-acclimated rye leaves

Electron probe X-ray microanalysis was used to analyse the effects of sub-zero temperatures on K⁺ distribution in compartments within non-acclimated and cold acclimated rye (Secale cereale L. cv Voima) leaf cells and to evaluate membrane leakage of ions caused by freezing-injury. The specimens were rapidly frozen from growing temperatures and from two different sub-zero temperatures (LT₅₀ and LT₁₀₀) to which the leaves had already been slowly cooled. Measurements were made in the cytoplasm, vacuole and cell walls in freeze-substituted mesophyll cells. At ambient temperatures, the mean K⁺ concentration in the cytoplasm (100 mol m^?3) differed significantly from that of the vacuole (49 mol m^?3) in the non-acclimated (NA) cells, while in cold acclimated (A) cells, the concentrations were similar (109 vs 93 mol m^?3, respectively). At LT₅₀ temperatures, the K⁺ concentration in NA-cells decreased significantly in the cytoplasm (59 mol m^?3) but increased in the cell walls. In the A-cells, on the other hand, the mean K⁺ concentration increased significantly (about three-fold) in all major compartments. At LT₁₀₀ temperatures, K⁺ concentrations in the cytoplasm and cell walls decreased when compared with corresponding LT₅₀ values in the A-cells but increased in the NA-cells. The increased potassium concentration in the cytoplasm of A-cells at LT₅₀ temperature is compatible with the observed cell shrinkage and an absence of plasma membrane damage. The decreased potassium concentration in the cytoplasm of NA-cells at LT₅₀ temperature is compatible with the slight cell shrinkage and suggests that the plasma membrane in these cells shows increased permeability due to freeze injury.     

A genetic linkage map of rye (Secale cereale L.)

 A genetic linkage map of rye composed of 91 loci (88 RFLP, two morphological and one isozyme markers) has been developed using two reciprocal crosses. The RFLP loci covering all seven chromosomes were detected by a selection of rye, wheat, barley and oat cDNA and genomic DNA probes. The level of polymorphism was dependent on the source of the clones, with a ranking of rye>wheat>barley>oat. Distorted segregations were detected in linkage groups of chromosomes 1R, 4R, 5R and 7R. When the recombination of the two reciprocal crosses was compared, no systematic increase or decrease in one or the other direction was observed suggesting that a combination of populations of reciprocal crosses is possible. Received: 5 August 1997/Accepted: 2 September 1997     

Chromosomal location of PCR fragments as a source of DNA markers linked to aluminium tolerance genes in rye

 To identify and locate rye DNA sequences homologous to three wheat c-DNAs (wali1, wali2 and wali5) whose expression is induced by aluminium (Al) stress, we designed three pairs of specific primers. They were used in the amplification of genomic DNA from wheat-rye disomic addition lines. The wali2 pair of primers amplified a 878-bp rye DNA fragment (rali2) located on chromosomes 4R and 7R that showed 79.37% homology with the corresponding wheat c-DNA. RAPD fragments were also used as genetic markers. We located 22 different RAPDs distributed on 11 different rye chromosome arms using wheat-rye disomic and ditelocentric addition lines. Thirteen of these markers were located on the chromosomes 3R, 4R and 6R, which also carry aluminium-tolerance genes. The OPA08 ₄₁₅ and OPR01 ₆₀₀ RAPD markers, located on the 6RL and 6RS chromosome arms, respectively, were converted to SCAR markers (SCA08 ₄₁₅ and SCR01 ₆₀₀ ) and linked to Alt1 gene (SCR01 ₆₀₀ -2.1 cM-Alt1-33.5 cM-SCA08 ₄₁₅ ). We propose that the chromosomal location of RAPDs and SCARs using wheat-rye addition lines is a source of DNA markers linked to aluminium-tolerance loci and offers a valuable strategy in marker-assisted selection for the introgression of tolerance genes in wheat. Received: 9 June 1997 / Accepted: 19 September 1997     

Rearrangement of the Cytoskeleton in Triticum aestivum Cells during Cold Hardening and after Treatment with Abscisic Acid

Immunocytochemical study of the basic characteristics of the tubulin and actin cytoskeleton (total content, orientation, structure, and stability) was performed for various root zones of the seedlings of winter wheat cultivars contrasting in their freezing tolerance. Plant cold hardening (3°C, 7 days) and ABA treatment (30 M, 3 days) increased the stability of tubulin microtubules (MT), that is, reduced the depolymerizing action of oryzalin in vivo. However, the mechanisms of hardening and ABA stabilizing action on the cytoskeleton were different: low temperature enhanced spatial MT aggregation and resulted in the formation of a dense network of thick MT bundles, whereas ABA reduced the content of tubulin components and induced microfilament (MF) depolymerization. Most pronounced temperature- and ABA-induced cytoskeleton changes were observed in the differentiation zone, which indicates an important role of this root zone in plant adaptation and development of root freezing tolerance. Low temperatures reduced the hormonal effect on the structural arrangement and stability of MT and MF in wheat cultivars of high and moderate freezing tolerance but increased hormonal effects in the slightly tolerant cultivar. MF depolymerization and an increase in the proportion of stable MT are supposed to be a necessary condition for seedling growth retardation after their treatment with ABA and for seedlings at the initial phase of their adaptation to low temperature. At the final phase of cold hardening, some growth acceleration is evidently determined by the accumulation of highly labile MT and greater actin polymerization.     

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     

The effect of photoperiod and temperature on growth and frost resistance of winter rye root systems

Root growth, development and frost resistance were examined in winter rye ( Secale cereale L. cv. Puma) plants grown under 6 combinations of temperature and photoperiod (20/16°C or 5/3°C, day/night; 8, 16- or 24-h days). Overall root system growth is influenced by the interaction of temperature and photoperiod. Maximum shoot growth occurs at a 24-h photoperiod in 20°C plants and at a 16-h photoperiod in 5°C plants, and is correlated in both treatments with a high root:shoot ratio. Frost resistance of rye roots is affected by short photoperiods in 2 ways. First, short photoperiod and low temperature delay production of new adventitious roots so that newly developing roots are not exposed to freezing temperatures. Second, short photoperiod alone can induce several degrees of frost tolerance in existing roots during the lag phase of growth. Low temperature alone does not decrease the rate of dry weight accumulation in rye root systems, but cold temperature does retard developmental processes within the roots. Rye roots grown at 5°C develop first order lateral roots, differentiate metaxylem vessels and suberize endodermal cell walls more slowly than roots grown at 20°C.     

Growth response and phosphorus uptake of rye with long and short root hairs: Interactions with mycorrhizal infection

Plant growth and phosphorus (P) uptake of two selections of rye (Secale cereale L.) differing in length of root hairs, in response to mycorrhizal infection were investigated. Rye plants with short root hairs (SRH) had a greater length of root infected by Glomus intraradices (up to 32 m pot^–1) than those with long root hairs (LRH) (up to 10 m pot^–1). Application of P decreased the percentage of root length infected in both selections. In low-P soil, mycorrhizal infection increased shoot and root P concentration, especially in LRH plants. Generally, LRH had higher shoot dry weight than SRH plants. P uptake was increased both by LRH and by mycorrhizal infection. Differences in specific P uptake and P utilization efficiency between SRH and LRH plants were observed in non-mycorrhizal plants. With low P supply, P utilization efficiency (dry matter yield per unit of P taken up) of LRH plants increased with time. However, mycorrhizal infection reduced P utilization efficiency, particularly of SRH plants. SRH plants, which were agronomically less efficient (i.e. low dry matter yield at low P supply) were more responsive to either mycorrhizal infection or P addition than the LRH plants. No interaction was observed between mycorrhizal infection and root hair length.     

Detection of quantitative trait loci on chromosome 5R of rye (Secale cereale L.)

 Progenies of an F₂ mapping population were analyzed for quantitative traits to detect QTLs by using marker information from F₂ plants for chromosome 5R. The mapping population was segregating for the major dwarfing gene Ddw1 and the gene Hp1 for hairy peduncle. The only QTL determining plant height was located between HP1 and Ddw1 on the distal part of chromosome 5RL. At the same position a QTL for peduncle length was found, and this trait was closely related to plant height (r=0.895). Since Hp1 and Ddw1 are dominant marker loci, no dominance effect could be estimated. The QTLs for spike length and the number of florets were located near the centromere on 5RL. These two traits were correlated with r=0.824 and showed partial dominance, but these traits were not correlated to plant height and peduncle length. Homoeologous relationships between the QTLs mapped for the first time in rye and those mapped in other Triticeae members are discussed. Received: 8 June 1998 / Accepted: 8 October 1998     

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     

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     

A comparative study of the fine structure of coleorhiza and root cells during the early hours of germination of rye embryos

Summary The sequences of changes which occur in the fine structure of root and coleorhiza cells of the rye embryo during the first 9 hours of germination are described. Quiescent cells from both tissues characteristically possess no vacuole, a cytoplasm densely packed with ribosomes, lipid droplets largely confined to a peripheral position, a greatly reduced endomembrane system, mitochondria with few cristae and nuclei in which the heterochromatin is condensed. Following imbibition the structure of root cells is elaborated slowly. Microtubules and dictyosomes appear, followed by the development of mitochondrial cristae and endoplasmic reticulum and the dispersion of lipid droplets. A similar pattern of events occurs within coleorhiza cells but at a much enhanced rate. By 6 hours the endomembrane system is highly organized but by 9 hours it has largely disappeared. These observations are discussed in relation to the penetration of the root through the coleorhiza.     

Phenylpropanoid compounds in primary leaf tissues of rye (Secale cereale). Light response of their metabolism and the possible role in UV-B protection

The present study was undertaken in order to investigate the suitability of certain markers for UV plant response. In addition, we attempted to link the internal tissue distribution of specific UV-absorbing compounds to profiles of radiation gradients within intact primary rye leaves ( Secale cereale L. cv. Kustro). Etiolated rye seedlings irradiated with low visible light (LL) and/or UV radiation were used to study enzyme activities of the two key enzymes, phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), together with the tissue-specific accumulation of soluble phenylpropanoid products. Plants grown under relatively high visible light (HL) with or without supplementary UV-B radiation were used for further characterization. Apparent quantum yield and fluorescence quenching parameters were monitored to assess potential physiological changes due to UV-B exposure in HL-grown seedlings. A quartz fibreoptic microprobe was used to characterize the internal UV-B gradient of the leaf. The response of the phenylpropanoid metabolism to UV radiation was similar in primary leaves of both etiolated and HL-treated green plants. The epidermis-specific flavonoids together with CHS activity turned out to be suitable markers for assessing the effect of UV on the phenolic metabolism. The functional role of phenylpropanoid compounds was strongly implicated in protecting rye from UV-B radiation.     

RFLP-based mapping of three mutant loci in rye (Secale cereale L.) and their relation to homoeologous loci within the Gramineae

 Three mutant loci of rye determining absence of ligules (al), waxless plant (wa1) and waxy endosperm (Wx) characters were mapped in a single F₂ population, comprising 84 individual plants. The three loci could be clearly tagged in relation to 7 (al on chromosome 2R), 4 (wa1 on chromosome 7R) or 6 (Wx on chromosome 4R) RFLP markers. The mapping data are compared with existing data for homoeologous regions containing equivalent mutants of wheat, barley, rice and maize. It is shown that the loci analysed are highly conserved across the cereal species, including rye. Received: 14 March 1997 / Accepted: 21 March 1997