Leaf senescence in a non-yellowing mutant of Festuca pratensis: implications of the stay-green mutation for photosynthesis, growth and nitrogen nutrition

Mutation of the nuclear gene sid disables chlorophyll degradation during leaf senescence in the pasture grass Festuca pratensis. This study investigated the effect of the mutation on photosynthesis and on leaf and whole plant growth under a range of nitrogen regimes. When plants were cultivated in a static hydroponic system, the chlorophyll content of fourth leaves of the stay-green mutant Bf993 remained virtually unchanged from full expansion to complete senescence, while tissue of the wild-type (cv. Rossa) became completely yellow. The retention of chlorophyll in Bf993 was not associated with maintenance of photosynthetic activity as shown by rates of light-saturated CO₂ fixation and apparent quantum efficiency. Higher levels of total N in senescing leaves of Bf993 than in Rossa indicated reduced nitrogen remobilization in the mutant. When using a range of [NH₄NO₃], dry matter production and tillering Mere lower for Bf993 at all but the highest [NH₄NO³, which was supra-optimal for the wild type. In contrast to the static system, where fluctuations in N supply occurred, growth and [NO₃^?] uptake were similar in mutant and wild type when [NO₃^?] was continuously maintained by a flowing solution culture system. The results are discussed in relation to the role of N supply and the effect of the stay-green mutation on N recycling.     

Anther Culture of Lolium temulentum, Festuca pratensis and Lolium x Festuca Hybrids. II. Anther and Pollen Development In Vivo and In Vitro

The various pathways of pollen development were investigatedin cultured anthers of Lolium temulentum, Festuca pratensisand the L. multiflorum x F. pratensis hybrid ‘Elmet’.In all three, development from the vegetative cell was the predominantpathway of pollen callus development. However, there were characteristicdifferences in the behaviour of the generative cell. In L. temulentumit remained attached to the pollen wall and degenerated, whereasin F. pratensis it divided. In ‘Elmet’ it detachedfrom the pollen wall and remained undivided. Both polarizedand unpolarized partitioned calluses were observed. Developmentof the fusion product of the vegetative and generative nucleiwere also observed in anthers of L. temulentum. Anomalous grainswere not found to be major source of pollen calluses. Sections of anthers of L. temulentum were used to investigatethe origin of S pollen grains, the small pale-staining grainswhich denote pollen dimorphism. Such grains form out of contactwith the tapetum and are therefore determined before or duringmeiosis (i.e. before harvest of anthers for culture). Sectionswere also used to demonstrate the influence of the durationof pretreatment on the development of the middle layer of theanther wall. Festuca pratensis, Lolium temulentum, Lolium x Festuca, anther culture, haploid, microspore, pollen     

Anther Culture of Lolium temulentum, Festuca pratensis and Lolium x Festuca Hybrids. I. Influence of Pretreatment, Culture Medium and Culture Incubation Conditions on Callus Production and Differentiation

This study was conducted with Lolium temulentum, Festuca pratensis,and the two hybrids L. multiflorum x F. pratensis ‘Elmet’and L. perenne x F. pratensis ‘Prior’. In a comparisonof various durations (7–42 d) of pretreatment at 4 or7 °C the highest yield of microspore-derived callus of L.temulentum was obtained after pretreatment of spikes at 7 °Cfor 28 d, conditions which also proved optimal for panicle pretreatmentwith F. pratensis. For ‘Elmet’, durations of 21–42d were optimal, and for ‘Prior’ the responses tendedto decline with increasing duration. In L. temulentum addition of charcoal (1–2 g l^–1)to medium containing 2, 4-D and KN wa     

Presence of Tyloses at the Blade/Sheath Junction in Senescing Leaves of Lolium temulentum L.

Aspects of age-related changes occurring in the blade/sheathjunction of leaves ofLolium temulentum L. were studied usingautoradiography, light and transmission electron microscopy.With increasing leafage the apical transport of sheath-appliedradioactive amino acids was retarded at the sheath side of theblade/sheath junction. An anatomical study revealed the presenceof tyloses in the protoxylcm lacunae of vascular bundles atthis region in senescing leaves. The possible involvement oftyloses in grass leaf senescence is discussed.     

Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization

Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum × F. pratensis (2n=4x=28) with L. multiflorum (2n=2x=14), the technique identified chromosomes with interspecific recombination. Also, in an introgressed line of L. multiflorum which was homozygous for the recessive sid (senescence induced degradation) allele from F. pratensis, a pair of chromosome segments carrying the sid gene could be identified, indicating the suitability of GISH in showing the presence and location of introgressed genes. By screening backcross progeny for the presence of critical alien segments and the absence of other segments the reconstitution of the genome of the recipient species can be accelerated.     

Leaf structure and translocation of dry matter in a C3 and a C4 grass

Summary Dry weight analyses and ¹⁴CO₂ were used to study translocation in leaves of the C₃ grass Lolium temulentum L. and the C₄ grass Panicum maximum Jacq. and the results related to the distribution and amount of phloem in the lamina. The rate of specific mass transfer rose from the tips to the bases of leaf blades, in both species high rates were recorded. Major veins were responsible for the bulk of longitudinal translocation and minor veins were important in collecting and loading photosynthate. Transverse veins stored ¹⁴C-assimilate and may have coordinated the functioning of the longitudinal veins. The bearing of the results on the mechanism of translocation is discussed.     

The host-plant relationships of apterous virginoparae of the grass aphid Metopolophiurn festucae cerealium

Metopolophium festucae cerealium has at various times caused damage to both grass and cereal crops. Despite this, little work has been done on the relative susceptibility of different grass and cereal cultivars to this aphid. The present work showed that for the cultivars examined the most common ley pasture species (Lolium perenne and Lolium multiflorum) exhibited relatively low levels of antibiotic resistance to M. f. cerealium and that wheat is more susceptible than any of the grass species tested. This enigma, in view of the aphid's low pest status on wheat, is discussed. The resistance in the grasses was greater at the stem elongation stages than at the seedling stage but the ranking was consistent between growth stages. M. f. cerealium exhibited a preference for feeding on emerging and senescing leaves of 10-wk-old grass but on wheat it distributed itself evenly between all available green leaves.     

Oligosaccharide intermediates of fructan synthesis in Lolium temulentum

Evidence is presented that fructan accumulation in leaves of Lolium temulentum plants grown at 5° proceeded via the synthesis of trisaccharide intermediates. Studies on the oligosaccharide components of this tissue indicated that the major intermediate was probably 1^F-fructosyl sucrose (isokestose) but that two distinct series of oligofructosides could be isolated. One of these had chromatographic properties identical to the 1,2-linked inulin series from Helianthus tuberosus. The relationship of this synthetic pattern to the structure of grass fructans and their accumulation in other species is discussed.     

Phylogeographical history of the widespread meadow fescue (Festuca pratensis Huds.) inferred from chloroplast DNA sequences

Aim Here we explore the variation in chloroplast DNA (cpDNA) in a widespread Eurasian diploid forage grass, meadow fescue (Festuca pratensis Huds.), to address its phylogeographical history. In particular, we aim to answer whether the post‐glacial migration routes of meadow fescue are associated with the spread of agriculture or concurrent with well‐documented natural migration pathways from glacial refugia. Location A total of 56 Eurasian accessions of F. pratensis were analysed, representing the entire native distribution area as well as non‐native areas in northernmost Europe. Methods Based on initial sequencing of 10 non‐coding cpDNA regions, three regions were sequenced for all F. pratensis accessions. For reference, three closely related species [the diploid Lolium perenne L. and the polyploids Festuca arundinacea Schreb. and Festuca gigantea (L.) Vill.] were also sequenced, as well as the more distantly related Festuca ovina L. Divergence times were estimated assuming a simple molecular clock, calibrated using a previously published estimate of 9 Myr for the divergence between fine‐leaved (F. ovina) and broad‐leaved fescues (F. pratensis, F. arundinacea and F. gigantea). Results Limited, but geographically structured, cpDNA variation was observed in F. pratensis. Three haplotypes, estimated to have diverged 0.16 Ma, were identified: one western European (A), one with a wide eastern distribution from central‐eastern Europe into Asia (B) and one Caucasian (C). The haplotypes of the polyploids and L. perenne were estimated to have diverged from haplotype A in F. pratensis 0.8–1.3 Ma. Main conclusions We found no definite evidence for migration of the diploid F. pratensis associated with the spread of agriculture from the Fertile Crescent after the last glaciation. The distinct geographical structuring of the present‐day variation in cpDNA can rather be explained by northwards expansion of the western haplotype from an Iberian refugium, expansion of the eastern haplotype from an unlocated (south‐)eastern refugium and glacial survival without subsequent expansion from a Caucasian refugium. The high level of cpDNA divergence observed between this diploid and the polyploids which have probably been derived from it may suggest that the very low level of cpDNA variation in the diploid is caused by a recent bottleneck. Today, F. pratensis is widespread in the open agricultural landscape but appears otherwise confined to naturally open habitats such as river banks, and its populations may have been decimated when dense forests dominated in the previous interglacial.     

Chloroplast DNA variation in the grass tribe Festuceae

Summary Six grasses, Hordeum sativum, Dactylis glomerata, Festuca arundinacea, F. pratensis, F. rubra and Lolium multiflorum were subjected to chloroplast DNA analysis based on restriction endonuclease digestion fragments and end labeling with ³⁵S nucleotides. This method is compared with others in general use. The results indicate that Lolium multiflorum is closely affiliated with Festuca pratensis and F. arundinacea; in fact much closer than F. rubra is to any of them.     

Relationship between Contents of Chlorophyll (a+b) (SPAD values) and Nitrogen of Some Temperate Grasses

In a field experiment the chlorophyll (a+b) (SPAD readings) and nitrogen contents of three grass species (Festuca arundinacea Schreb., Lolium perenne L., and Lolium multiflorum Lam.) and three intergeneric hybrids of Festuca pratensis Huds. × Lolium multiflorum Lam. and Festuca arundinacea Schreb. × Lolium multiflorum Lam. were measured. Close relationships were found between SPAD readings and nitrogen leaf content (r ² = 0.873^** 0.491^** and 0.938^**) for the 1^st, 2^nd, and 3^rd cut, respectively. SPAD readings and N contents were closely correlated (r = 0.836^**) confirming that SPAD measurements could be used in grass selection and/or breeding for high N concentration in herbage. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of caffeine,different fixation regimes and low temperature on microtubules in the cells of higher plants

Caffeine, (1:3:7-tri-methyl-xanthine), either as a prefixation treatment or included with glutaralde-hyde as the primary fixative, destroys or disorganises the microtubules associated with the formation of secondary walls in fibres from the flowering stem of the grass Lolium temulentum L. There is no observable effect of caffeine treatment on the microtubules associated with primary wall formation in collenchyma and young fibres from L. temulentum or in root cap cells of Zea mays L. and Phaseolus vulgaris L. The microtubules associated with primary wall formation are destroyed by cold treatment but not those associated with secondary wall formation. Tannic acid included in the fixative shows the microtubules associated with secondary wall formation in fibres of L. temulentum to be composed of 13 subunits. Treatment with lanthanum hydroxide does not stain the core or the halo of the microtubules.Abbreviation PIPES Piperazine N-N- bis 2 ethanol sulphonic acid The Grassland Research Institute is financed through the Agricultural Research Council     

Probing meiosis in hybrids of Lolium (Poaceae) with a discriminatory repetitive genomic sequence

A moderately repetitive genomic DNA sequence (designated pLPBB2-123) derived from Lolium perenne L. (Poaceae) is considerably more abundant in the genome of this species than in that of the closely related L. temulentum. The repetitive sequence probe is clearly able to discriminate between the genomic DNA of both species in Southern analysis, and effectively ’paints’ only the chromosome set of L. perenne in diploid and triploid hybrids with L. temulentum. Fluorescence in situ hybridisation of this sequence onto homoeologous chromosomes during meiosis I of the hybrids shows that the sequence is evenly distributed along all of the chromosomes of L. perenne and appears to have little effect on the structural integrity or recombination potential of hybrid bivalents. Discrimination between chromatin of different parental origin in hybrid bivalents shows for the first time a progressive relaxation of relational coiling of homoeologues throughout meiotic prophase. It also highlights structural irregularities that can now be unequivocally assigned to the longer chromosomes of L. temulentum. The advantages of the use of specific differentially amplified sequences instead of whole genome probes are discussed within the context of introgression breeding programmes within the Lolium/Festuca complex. Received: 14 December 1999; in revised form: 15 February 2000 / Accepted: 15 February 2000     

Light-induced Increase in Sucrose Phosphate Synthetase Activity in Leaves of Lolium temulentum

Mature leaves of Lolium temulentum L. were assayed for sucrosephosphate synthetase activity at different times during thephotoperiod. There was a rapid increase in activity at the onsetof illumination which was not observed in leaves maintainedin darkness. The activity prior to illumination was insufficientto catalyse the rates of sucrose synthesis observed in illuminateddetached leaves; after 15 min illumination the two processeswere of similar magnitude. Lolium temulentum L., darnel, sucrose phosphate synthetase, enzyme activity, light, sucrose, starch     

Leaf Development in Lolium temulentum L.: Progressive Changes in Soluble Polypeptide Complement and Isoenzymes

Ougham, Helen J., Jones, Thomas W. A. and Evans, Mair LL. 1987.Leaf development in Lolium temulentum L.: progressive changesin soluble polypeptide complement and isoenzymes.—J. exp.Bot. 38: 1689–1696. The spectrum of soluble polypeptides extracted from segmentsof the developing 4th leaf of Lolium temulentum simplified withincreasing distance from the leaf base. Most of the metabolicallyimportant isoenzymes analysed also exhibited gradients of activitywith respect to distance from the base, and in some cases twoor more contrasting gradients were observed for a given enzyme. Key words: Gradients, isoenzymes, leaves, Lolium temulentum,, soluble polypeptides     

Darkness promotes flowering in the absolute long-day requiring plant, Lolium temulentum L. Ceres

Vegetative plants of the long-day grass Lolium temulentum L.Ceres were exposed to threshold long days or light breaks. Protracteddarkness given just afterwards clearly promoted flowering andwas weakly inductive on its own. The promotive effect of darknesswas restricted to floral induction since further apical developmentwas weak. Key words: Lolium temulentum, flowering, photoperiodism, darkness     

Temperature and the translocation of photosynthate through the leaf of Lolium temulentum

Summary Localized cooling of a 2 cm length of the leaf blade of the grass Lolium temulentum to 0° C, had only a small effect on the mass transfer of assimilates from the distal part of the leaf. There was no evidence of a reduction in the velocity of movement of ¹⁴C-labelled assimilates through the low temperature zone, and the retention of assimilates along the pathway of movement was reduced by low temperature. In contrast to the longitudinal movement, lateral movement of ¹⁴C, induced by steam killing sections of the leaf, was extremely sensitive to temperature and showed no sign of acclimatization, even after 3 days at low temperature. These results suggest that, although loading sugars into the transport system was sensitive to temperature, the movement of assimilates was not directly controlled by metabolic processes along the pathway.     

Availability of cadmium and zinc accumulated in the leaves of Thlaspi caerulescens incorporated into soil

When grown on contaminated soil, hyperaccumulator plants contain high concentrations of metals which may return to the soil after senescence. This work was undertaken to assess the availability of Cd and Zn associated to the leaves of the hyperaccumulator Thlaspi caerulescens after incorporation into an uncontaminated soil. A Zn- and Cd- accumulator population of T. caerulescens was grown on a Cd- and Zn- contaminated soil previously labelled with ¹⁰⁹Cd. Leaves (TCL) were harvested, dried, ground and incorporated into the soil at a rate of 2.07 mg Cd kg⁻¹ and 51.9 mg Zn kg⁻¹. Then a pot experiment was conducted for 3 months with rye grass (Lolium perenne) and T. caerulescens. Rye grass was harvested monthly and T. caerulescens at the end of the experiment. Plant biomass was measured, along with the concentration of Cd, Zn and ¹⁰⁹Cd. Results showed that water-extractable metals in TCL were 69% for Zn and 33% for Cd. Addition of TCL to soil, depleted growth of rye grass, and improved that of T. caerulescens. At harvest, concentrations of both metals were increased in plants by TCL. Concentrations of Cd in rye grass increased with the cut number, while that of Zn decreased slightly. Rye grass extracted 1.6% of the total Cd and 0.9% of the total Zn, and T. caerulescens extracted up to 22.4% of the Cd and 7% of the Zn. About 94% of the Cd in rye grass and 86% in T. caerulescens was derived from TCL. In conclusion, metals associated with leaves of the hyperaccumulator T. caerulescens were very mobile after incorporation into the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information

DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids. Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis, respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level. Chromosome mapping of these markers using a Lolium/Festuca substitution line revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolating the role of soil resources,defoliation, and interspecific competition on early establishment of the late successional bunchgrass Festuca campestris

Native grasslands are valued for biodiversity and supporting dormant season grazing, but are prone to invasion. In western Canada, revegetation of Festuca campestris grasslands may be hindered by Poa pratensis, an invasive grass. To determine the competitive interaction of these species during establishment, two greenhouse experiments were conducted where F. campestris seedlings were planted in monocultures or mixtures with P. pratensis. The first experiment used equal‐aged (3‐month old) seedlings of both species, while the second experiment used unequal‐aged seedlings (4‐month‐old F. campestris and 2‐month‐old P. pratensis). Seedling performance was measured in response to manipulations of water and nitrogen, defoliation, and plant neighbor. While water and nitrogen reduced the biomass and vegetative reproduction (tillering) of F. campestris, exposure to P. pratensis most strongly limited the growth of F. campestris seedlings regardless of other treatments. More frequent and consistent decreases in F. campestris due to P. pratensis were observed in older F. campestris seedlings than younger seedlings. Defoliation also reduced the growth of F. campestris, and the added presence of P. pratensis during defoliation further enhanced these reductions in younger, equal‐aged bunchgrass seedlings. Overall, these results suggest that when restoring native F. campestris grasslands, early establishment may be improved by reducing the negative impacts of P. pratensis, and avoiding severe defoliation.