Long-day induction of flowering in Lolium temulentum involves sequential increases in specific gibberellins at the shoot apex

One challenge for plant biology has been to identify floral stimuli at the shoot apex. Using sensitive and specific gas chromatography-mass spectrometry techniques, we have followed changes in gibberellins (GAs) at the shoot apex during long day (LD)-regulated induction of flowering in the grass Lolium temulentum. Two separate roles of GAs in flowering are indicated. First, within 8 h of an inductive LD, i.e. at the time of floral evocation, the GA(5) content of the shoot apex doubled to about 120 ng g(-1) dry weight. The concentration of applied GA(5) required for floral induction of excised apices (R.W. King, C. Blundell, L.T. Evans [1993] Aust J Plant Physiol 20: 337-348) was similar to that in the shoot apex. Leaf-applied [(2)H(4)] GA(5) was transported intact from the leaf to the shoot apex, flowering being proportional to the amount of GA(5) imported. Thus, GA(5) could be part of the LD stimulus for floral evocation of L. temulentum or, alternatively, its increase at the shoot apex could follow import of a primary floral stimulus. Later, during inflorescence differentiation and especially after exposure to additional LD, a second GA action was apparent. The content of GA(1) and GA(4) in the apex increased greatly, whereas GA(5) decreased by up to 75%. GA(4) applied during inflorescence differentiation strongly promoted flowering and stem elongation, whereas it was ineffective for earlier floral evocation although it caused stem growth at all times of application. Thus, we conclude that GA(1) and GA(4) are secondary, late-acting LD stimuli for inflorescence differentiation in L. temulentum.     

Synaptonemal complex formation in hybrids of Lolium temulentum x Lolium perenne (L.)

Comparisons were made between two kinds of tetraploids derived from the hybrid Lolium temulentum x L. perenne. One hybrid behaves like an autotetraploid with multivalents at first metaphase of meiosis in pollen mother cells. The other behaves like an allotetraploid, in which pairing at first metaphase is restricted to bivalents comprised of strictly homologous chromosomes. The diploidisation of the latter form is controlled by determinants located on both the normal, A chromosomes and on supernumary B chromosomes. Reconstruction of synaptonemal complexes and their elements, from serial sections through pollen mother cell nuclei examined under the electron microscope, reveals that at zygotene pairing in both forms results in multivalent formation involving non-homologous as well as homologous chromosomes. The mechanism responsible for the diploidisation is, therefore, not based on a restriction of pairing at early meiosis to homologous chromosomes but on a correction or transformation of the multivalent chromosome associations to bivalents subsequent to zygotene. The transformation is not completed until late pachytene. In the multivalent-forming tetraploid a maximum of four chromosomes are associated at first metaphase. Yet configurations of a higher valency are found at zygotene. There is, therefore, a partial transformation of multivalents even in this autotetraploid form which restricts configurations at metaphase I to homologous and homoeologous chromosomes only. In both hybrids some homologous bivalents are not the product of resolution of multivalents but result from two-by-two pairing from the beginning of zygotene.     

Enzymes of nitrogen mobilization in detached leaves of Lolium temulentum during senescence

During the senescence of Lolium temulentum leaf sections in the dark, asparagine and glutamine accumulated as the level of soluble protein declined. During the first 3–4 days after detachment, when the rate of protein loss was maximal, a four-fold increase in acid protease activity (EC 3.4.4.?) occurred. Subsequently this activity was replaced by proteases with a higher pH optimum. There was also a pronounced and continued activation of glutamate dehydrogenase (EC 1.4.1.2) during senescence. Glutamate pyruvate transaminase (EC 2.6.1.2), benzoylarginine-p-nitroanilide hydrolase (EC 3.4.?.?) and leucyl-p-nitroanilide hydrolase (EC 3.4.1.1) declined from high initial activities after 3–4 days. Glutamate oxaloacetate transaminase (GOT, EC 2.6.1.1) was fairly stable although a marked increase occurred in the activity of one of two major GOT isoenzymes over the first two days. Glutamine synthetase (EC 6.3.1.2) was highly active in non-senescent leaves but fell sharply during the first three days of senescence. Little asparagine synthetase (EC 6.3.1.1) was detected. The role of these enzymes in the nitrogen metabolism of senescent detached leaves is discussed.     

The Developmental Anatomy of Leaves in Lolium temulentum

Measurements of the epidermal cells in Lolium temulentum andassessments of cell number indicate that differences in bladewidth are due mostly to variations in cell number, whereas changesin blade and sheath length result mainly from differences incell length. A marked increase in length of the flag-leaf aheath,however, was related to an increase in cell number. Considerable changes in the relative proportions of leaf bladeand sheath were observed in the flag leaf, and the leaves immediatelypreceding it, associated with inflorescence initiation. As oneconsequence of this the area of the flag leaf, the largest onthe shoot, is virtually constant under different environmentalcondi-tions. It is suggested that this aspect of correlateddevelopment is related to the nutrition of the panicle, sincein annual grasses such as the cereals the flag leaf may be responsiblefor producing up to 30 per cent. of the starch in the grain.     

Chromosome pairing in a Lolium temulentum X Lolium perenne diploid hybrid with a low chiasma frequency

Summary Despite an average difference of about 50% in DNA amount, homoeologous chromosomes pair effectively at first metaphase in the diploid interspecific hybrid between Lolium temulentum and Lolium perenne. However, in the presence of accessory B chromosomes and diploidising genes pairing at metaphase I is severely reduced. Reconstruction of serial electron micrographs through pollen mother cell nuclei show that synaptonemal complexes are formed at pachytene between not only homoeologous but also non-homologous chromosome segments resulting in multivalent formation. These associations are largely ineffective in terms of chiasma formation and degenerate at late pachytene. It is highly probable that the pairing determinants exercise their control on chromosome pairing largely by prohibiting the siting of crossovers in homoeologously paired chromosome segments.     

Changes in gene expression in the leaf of Lolium temulentum L. Ceres during the photoperiodic induction of flowering

Unifoliated plants of Lolium temulentum L. Ceres were induced to flower by a unique 24-h long day (LD) consisting of the extension of the regular 8-h short day (SD) (400 mol photons·m^–2·s^–1, fluorescence + incandescence) with incandescence at 10–15 mol photonsm ^–2·s^–1. The polyadenylated-RNA complement of leaf blade tissues was analysed at 4-h intervals during the photoperiod extension in LD vs. SD, by using two-dimensional polyacrylamide gel electrophoresis to resolve in-vitro-translated products. Of the 991 spots that were analysed, none appeared or disappeared during the inductive cycle, i.e. no qualitative effect of floral induction was detected, at any time. Sixty-eight spots were found whose intensity was influenced by lengthening of the photoperiod; 50 of them, i.e. ca. 5% of the population analysed, were affected before the end of the extension period and were thus potentially related to floral induction. Many of these RNAs were not quantitatively constant during a 24-h cycle in SD. Seven of them oscillated according to the light-on and the light-off signals, among which three seemed to be controlled by phytochrome since their relative amount increased under the standard light conditions but decreased under incandescence even faster than in darkness. The large majority of other RNAs varied with a timing that was not clearly driven by the alternation of light and darkness, indicating that genes related to the biological clock may be especially sensitive to the lengthening of the photoperiod. Furthermore, seven spots were observed that underwent a phase-shift in LD, which consisted, for six of them, of a phase advance of 4–8 h. The steady-state level of CAB mRNA was analysed because the CAB gene family (encoding the chlorophyll a/b-binding proteins of the light-harvesting complexes) is known to be controlled both by the biological clock and phytochrome. In SD, the level was high in the light and low in darkness; the fluctuation was conducted by a circadian rhythm. When plants were exposed to the inductive LD, the peak of mRNA accumulation that was expected according to the endogenous rhythmicity was abolished, possibly because of the change in light quality during the LD extension.Abbreviations CAB chlorophyll a/b-binding proteins of the light-harvesting complexes - 2D two-dimensional - LD(s) longday(s) - LDP(s) long day plant(s) - SD(s) short day(s) - SDP(s) short day plant(s) This work was supported by the University of Liège through the Action de Recherche Concertée (# 88/93-129). Some analyses were performed with the collaboration of Dr. H. Ougham, Institute of Grassland and Environmental Research, Aberystwyth, UK. The authors also want to thank Dr. F. Cremer (Max Planck Institute for Plant Breeding, Köln, Germany) for critical discussion of the results.     

The genetics of meiotic chromosome pairing in Lolium temulentum x Lolium perenne tetraploids

Summary The degree of preferential pairing of homologous chromosomes was estimated in a series of tetraploid hybrids of Lolium temulentum x Lolium perenne by means of cytological and genetic analyses. The correlations between the frequency of bivalents at first metaphase of meiosis in the hybrid tetraploids and the degree of preferential pairing calculated from the segregation pattern of isozyme alleles in a test cross was extremely high. The results showed clearly that suppression of heterogenetic pairing in these Lolium tetraploids is achieved by a genetic system involving the A chromosomes as well as the B chromosome system which has been known for some time. Certain similarities with the genetic system controlling pairing in polyploid wheats are discussed.     

Cloning and characterization of a salt stress-inducible small GTPase gene from the model grass species Lolium temulentum

A gene encoding a small guanosine triphosphate (GTP)-binding protein (smGTP) related to the Rab2 gene family of GTPases was identified during the analysis of a salt stress suppression subtractive hybridization (SSH) expression library from the model grass species Lolium temulentum L. (Darnel ryegrass). The smGTP gene was found to have a low-level constitutive expression and was strongly induced by salt stress in root, crown and leaf tissues. The expression pattern of the smGTP gene was compared against two additional stress genes identified in the SSH expression library, the well-characterized dehydration stress tolerance gene, delta 1-pyrroline-5-carboxylate synthetase (P5CS) encoding for a key enzyme in proline biosynthesis, and the cold response gene COR413. The genes were analyzed for their response to salinity as well as their responses to 7 different forms of abiotic stress in L. temulentum plants. The smGTP gene displayed an expression pattern similar to the P5CS gene, suggesting a role in dehydration stress. In contrast, the COR413 gene was found to be up-regulated in response to all stresses tested and has utility as a general stress marker in grass plants.     

Acclimation of Lolium temulentum to enhanced carbon dioxide concentration

Acclimation of single plants of Lolium temulentum to changing[CO₂] was studied on plants grown in controlled environmentsat 20°C with an 8 h photoperiod. In the first experimentplants were grown at 135 µ;mol m^–2 s^–1 photosyntheticphoton flux density (PPFD) at 415µl l^–1 or 550µll^–1 [CO₂] with some plants transferred from the lowerto the higher [CO₂] at emergence of leaf 4. In the second experimentplants were grown at 135 and 500 µmol m^–2 s^–1PPFD at 345 and 575 µl l^–1 [CO₂]. High [CO₂] during growth had little effect on stomatal density,total soluble proteins, chlorophyll a content, amount of Rubiscoor cytochrome f. However, increasing [CO₂] during measurementincreased photosynthetic rates, particularly in high light.Plants grown in the higher [CO₂] had greater leaf extension,leaf and plant growth rates in low but not in high light. Theresults are discussed in relation to the limitation of growthby sink capacity and the modifications in the plant which allowthe storage of extra assimilates at high [CO₂]. Key words: Lolium, carbon dioxide, photosynthesis, growth, stomatal density     

Abiotic stresses activate a MAPkinase in the model grass species Lolium temulentum

Forage and turf grasses are utilized in diverse environments that expose them to a variety of abiotic stresses, however very little is known concerning the perception or molecular responses to these various stresses. In the model grass species Lolium temulentum, a 46kDa mitogen-activated protein kinase (MAPK) was activated in the leaf within 10min of exposing the roots to salt stress. When plants were subjected cold stress, no significant activation of the MAPK was observed. However, the 46kDa MAPK was rapidly activated in the leaves of plants within 3min of exposure to heat stress. Previously, mechanical wounding has been shown to rapidly activate a 46kDa and a 44kDa MAPK in L. temulentum. The wound activation of the MAPKs was delayed and diminished in plants undergoing cold treatment. In plants subjected simultaneously to 40°C and wounding, the activation of the 46kDa MAPK was enhanced. However if plants were subjected to heat and cold stress for more than 2h or exposed to 300mM NaCl for 24h prior to wounding, the wound activation of the 46kDa and a 44kDa MAPKs were significantly inhibited. These results suggest that the 46kDa MAPK plays a role in the response to various environmental stimuli.     

Gibberellin structure and florigenic activity in Lolium temulentum,a long-day plant

Structural requirements for florigenic activity among gibberellins (GAs) and GA derivatives, including several new ones, applied once to leaves of Lolium temulentum, were examined. The compounds were applied to plants kept either in non-inductive short days (SD) or exposed to one inductive long day (LD). Inflorescence initiation and stem-elongation responses were assessed three weeks later. Among the GAs used, the range in effective dose for inflorescence initiation was more than 1000-fold, but substantially less for stem elongation. Some GAs promoted both stem elongation and inflorescence initiation, some promoted one without the other, and some affected neither. The structural features enhancing florigenic activity were often different from those enhancing stem elongation. Except in the case of 2,2-dimethyl GA₄, a double bond in the A ring at either C-1,2 or C-2,3 was essential for high florigenic activity, though not for stem elongation. A free carboxy group was needed for both. Inflorescence initiation in Lolium was enhanced by hydroxylation at C-12, ?13 and ?15, whereas hydroxylation at C-3 reduced the effect on inflorescence initiation but increased that on stem elongation. A 12β-hydroxyl was more effective than the α epimer for inflorescence initiation whereas the reverse was true for stem elongation. Although such differential effectiveness of GAs for inflorescence initiation and for stem elongation could reflect differences in uptake, transport or metabolism, we suggest that it is indicative of specific structural requirements for inflorescence initiation.     

Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses, because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4–6 weeks of selection and transgenic plants were produced in 10–13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable tool for functionality tests of candidate genes in forage and turf grasses.