Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil

Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H₂ evolution in Ar:O₂ (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO₂ production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O₂ permeability decreased 50% within 5 hours in each species. A similar decrease in the O₂-saturated respiration rate (V_max) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V_max in each species. V_max may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O₂ inactivation of nitrogenase.     

Transformation of tobacco and birdsfoot trefoil by lupin leghemoglobin I cDNA clone

The full length cDNA clone of leghemoglobin I gene fromLupinus luteus was placed under dual promoter into the plant expression vector pCB1399 and the resulting vector (pCB1415) was transfered into theAgrobacterium strain LBA4404 (pAL4404). The binary system LBA4404 (pAL4404, pCB1415) was then used for transformation ofNicotiana plumbaginifolia andLotus corniculatus. In both species kanamycin-resistant plants have been selected and regenerated. The synthesis of LbI protein in transformed plants has not been shown.     

Isolation,culture and regeneration of protoplasts from birdsfoot trefoil (Lotus corniculatus)

A method was developed for rapid plant regeneration from protoplasts of birdsfoot trefoil (Lotus corniculatus L. cv. Leo). Green cotyledons from in vitro grown seedlings were preplasmolyzed in CPW salts containing 13% mannitol (CPW 13 M) for 1 h prior to the enzyme treatment. The enzyme formula consisted of 2% (w/v) Onozuka Cellulase R-10, 1% (w/v) Macerase and 0.1% (w/v) Pectolyase Y-23 in CPW 13 M. This method produced high yields of viable protoplasts after purification. The procedure is reproducible and takes approximately 2.5 months from protoplast isolation to plantlet establishment in a greenhouse. More than 100 plantlets were grown in soil. Two somaclonal variants, a chimeric plant for chlorophyll production and an albino cell line, have been obtained by this procedure.     

Effects of competitive stress on vegetative growth, storage, and regrowth after defoliation in Phleum pratense

The capacity for resource storage is important to population persistence in nonforaging plants with a consolidation strategy. In a competitive environment, selection may favor genetic variants with a well-developed ability to store resources for future vegetative growth or to regrow following herbivory. To determine the evolutionary potential for changes in vegetative growth, storage and regrowth after defoliation in response to competitive stress, half-sib families of the caespitose grass Phleum pratense were grown in pots in a glasshouse either alone or in competition with four individuals of Lolium perenne . They were defoliated after 16 wk and permitted to regrow for 8 wk. Production of new tillers, leaf area and leaf dry mass were recorded before and after defoliation; resource storage was estimated by the dry mass of stem bases. Tiller numbers, leaf area and mass, regrowth, and stem base mass were significantly reduced by competition; however, there was significant variation among families for many variables. A significant proportion of the variation in clipped and regrowth leaf mass, and in stem base mass, was due to a competition by family interaction. Storage in stem bases was correlated with total accumulated leaf mass in both control and competition groups. Genetic variation for competitive ability and the ability to regrow rapidly after defoliation exist in this P. pratense population. A well-developed ability to regrow following release from competitive or grazing pressures is likely to be a critical adaptation of caespitose grasses.     

Effects of sugar on vegetative development and floral transition in Arabidopsis

Although sugar has been suggested to promote floral transition in many plant species, growth on high concentrations (5% [w/v]) of sucrose (Suc) significantly delayed flowering time, causing an increase in the number of leaves at the time of flowering in Arabidopsis. The effect of high concentrations of Suc seemed to be metabolic rather than osmotic. The delay of floral transition was due to extension of the late vegetative phase, which resulted in a delayed activation of LFY expression. In addition, growth on low concentrations (1% [w/v]) of Suc slightly inhibited flowering in wild-type plants. This delay resulted from effects on the early vegetative phase. This inhibition was more pronounced in tfl1, an early flowering mutant, than in the wild type. Although 1% (w/v) Suc was reported to promote floral transition of late-flowering mutants such as co, fca, and gi, floral transition in these mutants was delayed by a further increase in Suc concentration. These results suggest that sugar may affect floral transition by activating or inhibiting genes that act to control floral transition, depending on the concentration of sugars, the genetic background of the plants, and when the sugar is introduced. Growth on 1% (w/v) Suc did not restore the reduced expression levels of FT and SOC1/AGL20 in co or fca mutants. Rather, expression of FT and SOC1/AGL20 was repressed by 1% (w/v) Suc in wild-type background. The possible effects of sugar on gene expression to promote floral transition are discussed.     

Analysis of quantitative traits connected with seed weight and flowering terms in birdsfoot trefoil (Lotus corniculatus L.) plants

The nature of inheritance of characters connected with the seed size (seed mass) and quality of the shoots (germination energy, seed viability, general shoot length, shoot mass, dry weight of shoots) in Lotus corniculatus L. was analyzed. The investigations were carried out on plants of three varieties: the wild form from the Krasnodar Region and local forms MF1 and MF3. The correlation analysis was carried out. The pattern of inheritance in the terms of plant flowering and length of shoots were studied.     

Photosynthesis, N(2) fixation and taproot reserves during the cutting regrowth cycle of alfalfa under elevated CO(2) and temperature

Future climatic conditions, including rising atmospheric CO₂ and temperature may increase photosynthesis and, consequently, plant production. A larger knowledge of legume performance under the predicted growth conditions will be crucial for safeguarding crop management and extending the area under cultivation with these plants in the near future. N₂ fixation is a key process conditioning plant responsiveness to varying growth conditions. Moreover, it is likely to increase under future environments, due to the higher photosynthate availability, as a consequence of the higher growth rate under elevated CO₂. However, as described in the literature, photosynthesis performance is frequently down-regulated (acclimated) under long-term exposure to CO₂, especially when affected by stressful temperature and water availability conditions. As growth responses to elevated CO₂ are dependent on sink-source status, it is generally accepted that down-regulation occurs in situations with insufficient plant C sink capacity. Alfalfa management involves the cutting of shoots, which alters the source-sink relationship and thus the photosynthetic behaviour. As the growth rate decreases at the end of the pre-cut vegetative growth period, nodulated alfalfa plants show photosynthetic down-regulation, but during regrowth following defoliation, acclimation to elevated CO₂ disappears. The shoot harvest also leads to a drop in mineral N uptake and C translocation to the roots, resulting in a reduction in N₂ fixation due to the dependence on photosynthate supply to support nodule function. Therefore, the production of new shoots during the first days following cutting requires the utilization of reduced C and N compounds that have been stored previously in reserve organs. The stored reserves are mediated by phytohormones such as methyl jasmonate and abscisic acid and in situations where water stress reduces shoot production this potentially enables the enhancement of taproot protein levels in nodulated alfalfa, which may lead to these plants being in better condition in the following cut/regrowth cycle. Furthering our knowledge of legume performance under predicted climate change conditions will be crucial for the development of varieties with better adaptation that will achieve greater and more efficient production values. Furthermore, for this purpose it will be necessary to improve existing methodologies and create new ones for phenotype characterization. Such knowledge will provide key information for future plant breeding programs.     

Histochemical study of vegetative and floral bud formation in tobacco stem expiants

Primary explants from the inflorescence stem of tobacco and primary explants from the stem of vegetative plants, cultivatedin vitro under the same conditions, display different morphogenetic ability. The former give rise mostly to floral buds, whereas the latter exclusively to vegetative ones. Histological and histochemical analyses of both original andin vitro cultivated explants were made. They showed differences in chlorophyll content and alcohol dehydrogenase (AD) activity of the original explants reflecting their different metabolic status. Bud primordia were initiated in the superficial meristematic layer derived from epidermal tissues. Floral or prefloral apices were characterized by a strong AD activity in all cells of the meristem, while in vegetative apices AD activity was restricted to their uppermost parts. A high rate of procambium differentiation connected with leaf primordia formation was typical of vegetative buds. A higher concentration of glucose (5 %) enhanced cell division in explants, which is also correlated with a higher AD activity. The significance of vascular tissues for differentiation of vegetative buds is discussed. Presented at the International Symposium “Plant Growth Regulators” held on June 18-22, 1984 at Liblice, Czechoslovakia.     

Condensed tannins in the tissue culture of sainfoin (Onobrychis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.)

Two forage legumes, birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia Scop.), containing condensed tannins in their leaves and stems were used as source material to study condensed tannins in tissue culture. More protoplasts were isolated from mesophyll tissue of a low tannin-containing strain of birdsfoot trefoil than from a high tannin-containing strain, but more tannin-filled protoplasts were observed in the latter. Growth rates of leaf explant-derived callus tissue were greater for the high-tannin than for the low-tannin strain. In sainfoin, callus cultures from leaf explants produced numerous tannin-filled cells by 21 days. Explants from sainfoin cotyledons and roots, tissues which normally do not contain tannins, also formed callus with tannin-filled cells in 21 days but in almost every case, a cytokinin was required for tannin formation to occur. The occurrence of tannin-filled cells in callus from root and cotyledon explants was variable and genotype specific. These results show that endogenous tannins can affect protoplast isolation and possibly callus growth in birds-foot trefoil, and that the formation of condensed tannins in sainfoin callus culture can be influenced by a growth regulator.Abbreviations BAP benzylaminopurine - KIN kinetin - NAA naphthaleneacetic acid - PAR photosynthetically active radiation Contribution no. 920 of Agriculture Canada Research Station, 107 Science Cres., Saskatoon, Saskatchewan, Canada S7N OX2     

The systematic utility of floral and vegetative fragrance in two genera of nyctaginaceae

We examined relationships between fragrance and phylogeny using a number of approaches to coding fragrance data and comparing the hierarchical information in fragrance data with the phylogenetic signal in a DNA sequence data set. We first used distance analyses to determine which coding method(s) best distinguishes species while grouping conspecifics. Results suggest that interspecific differences in fragrance composition were maximized by coding as presence/absence of fragrance compounds and biosynthetic pathways rather than when quantitative information was also included. Useful systematic information came from both compounds and pathways and from fragrance emitted by both floral and vegetative tissues. The coding methods that emerged from the distance analyses as best distinguishing species were then adapted for use in phylogenetic analysis. Although hierarchical signal among fragrance data sets was congruent, this signal was highly incongruent with the phylogenetic signal in the DNA sequence data. Notably, topologies inferred from fragrance data sets were congruent with the DNA topology only in the most distal portions (e.g., sister group pairs or closely related species that had similar fragrance profiles were often recovered by analyses of fragrance). Examination of consistency and retention indices for individual fragrance compounds and pathways as optimized onto one of the most-parsimonious trees inferred from DNA data revealed that although most compounds were homoplastic, some compounds were perfectly congruent with the DNA phylogeny. In particular, compounds and pathways found in a few taxa were less homoplastic than those found in many taxa. Pathways that synthesize few volatiles also seem to have lower homoplasy than those that produce many. Although fragrance data as a whole may not be useful in phylogeny reconstruction, these data can provide additional support for clades reconstructed with other types of characters. Factors other than phylogeny, including pollinator interactions, also likely influence fragrance composition.     

Utilization of carbon and nitrogen reserves of alfalfa roots in supporting N2-fixation and shoot regrowth

Perennial legume such as alfalfa have the capacity to sustain shoot regrowth and some nodule N₂-fixation after removal (cutting) of shoots which contain practically all of the plant's photosynthetic capacity. The role of the roots in supporting these processes has not been fully described. Measurements were made of the nodules' responses to removal of shoots from 8-week-old seedlings in terms of N₂-fixation, as nitrogenase activity (NA) measured as acetylene reduction, dark CO₂ fixation, measured as in vitro phosphoenolpyruvate carboxylase (PEPC) activity, and total non-structural carbohydrate (NSC) content. These properties decreased and recovered in that sequence, which suggests that nodule NSC supported the substrate requirements of NA and PEPC immediately after cutting. The utilization and redistribution or root carbon and nitrogen, prelabeled with ¹⁴C and ¹⁵N, were also followed after cutting 8-week-old alfalfa seedlings. In the first 2 weeks of regrowth 12% of root C and 25% of root N were transferred for incorporation into new shoots. Up to 40% of the root C was used for plant respiration to support 28 days of shoot regrowth and N₂-fixation. The decline of N₂-fixation was slower after cutting and its minimum activity rose up 45% of pre-cut activity as root reserves were built up with plant age. Therefore, the stored reserves of nodulated roots play an important role in support of N₂-fixation after cutting.Contribution No. 1265 from Plant Research Center.Contribution No. 1265 from Plant Research Center.     

Nitrogen and methyl jasmonate induction of soybean vegetative storage protein genes

Vegetative storage protein (VSP) and VSP mRNA levels in soybean (Glycine max) leaves correlated with the amount of NH₄NO₃ provided to nonnodulated plants. The mRNA level declined as leaves matured, but high levels of N delayed the decline. This is consistent with the proposed role for VSP in the temporary storage of N. Wounding, petiole girdling, and treatment with methyljasmonate (MeJA) increased VSP mRNA in leaves 24 hours after treatment. The magnitude of the response depended on leaf age and N availability. N deficiency essentially eliminated the response to wounding and petiole girdling. MeJA was almost as effective in N-deficient plants as in those receiving abundant N. Inhibitors of lipoxygenase, the first enzyme in the jasmonic acid biosynthetic pathway, blocked induction by wounding and petiole girdling but not by MeJA. This supports a role for endogenous leaf jasmonic acid (or MeJA) in the regulation of VSP gene expression.     

Recovery of patients after four months or more in the persistent vegetative state.

A retrospective review was made of the case notes of 43 consecutive patients admitted to a unit specialising in the rehabilitation of people in the persistent vegetative state. Eleven of these patients regained awareness four months or more after suffering brain damage. The time to the first reported incidence of eye tracking was between four months and three years, and the time to the first response to command was between four and 12 months. Only one patient was eventually unable to communicate, six could use non-verbal methods of indicating at least a yes or no response, and four were able to speak. Six patients remained totally dependent while two became independent in daily activities. Four patients became independent in feeding, three required help, and four remained on gastrostomy feeding. Thus some patients can regain awareness after more than four months in a vegetative state, and, although few reach full independence, most can achieve an improved quality of life within the limitations of their disabilities. The recovery period is prolonged and may continue for several years. Even patients with profound brain damage should be offered the opportunity of a specialist rehabilitation programme.     

Mobilization of nitrogen reserves during regrowth of defoliated Trifolium repens L. and identification of potential vegetative storage proteins

Although it is well established that carbon reserves contributeto shoot regrowth of leguminous forage species, little informationis available on nitrogen reserves except in Medicaqo sativaL. and Trifolium subterraneum L. In this study, reserves werelabelled with ¹⁵N to demonstrate the mobilization of endogenousnitrogen from roots and stolons to regrowing leaves and newstolons during 24 d of regrowth in white clover (Thfolium repensL.). About 55% and 70%, respectively, of the nitrogen contentsof these organs were mobilized to support the regrowth of leaves.During the first 6 d, nitrogen in regrowing leaves came mainlyfrom N reserves of organs remaining after defoliation. Afterthese first 6 d of regrowth, most of the shoot nitrogen wasderived from exogenous nitrogen taken up while the contributionof nitrogen reserves decreased. After defoliation, the buffer-solubleprotein content of roots and stolons decreased by 32% duringthe first 6 d of regrowth. To identify putative vegetative storageproteins, soluble proteins were separated using SDS-PAGE ortwo-dimensional electrophoresis. One protein of 17.3 kDa instolons and two proteins of 15 kDa in roots seemed to behaveas vegetative storage proteins. These three polypeptides, initiallyfound at high concentrations, decreased in relative abundanceto a large extent during early regrowth and then were accumulatedagain in roots and stolons once normal growth was re-established. Key words: White clover, regrowth, ¹⁵N-labelled, vegetative storage proteins, electrophoresis     

Intra-population comparison of vegetative and floral trait heritabilities estimated from molecular markers in wild Aquilegia populations

Measuring heritable genetic variation is important for understanding patterns of trait evolution in wild populations, and yet studies of quantitative genetic parameters estimated directly in the field are limited by logistic constraints, such as the difficulties of inferring relatedness among individuals in the wild. Marker-based approaches have received attention because they can potentially be applied directly to wild populations. For long-lived, self-compatible plant species where pedigrees are inadequate, the regression-based method proposed by Ritland has the appeal of estimating heritabilities from marker-based estimates of relatedness. The method has been difficult to implement in some plant populations, however, because it requires significant variance in relatedness across the population. Here, we show that the method can be readily applied to compare the ability of different traits to respond to selection, within populations. For several taxa of the perennial herb genus Aquilegia, we estimated heritabilities of floral and vegetative traits and, combined with estimates of natural selection, compared the ability to respond to selection of both types of traits under current conditions. The intra-population comparisons showed that vegetative traits have a higher potential for evolution, because although they are as heritable as floral traits, selection on them is stronger. These patterns of potential evolution are consistent with macroevolutionary trends in the European lineage of the genus.     

Palatability in sheep and in vitro nutritional value of dried and ensiled sainfoin (Onobrychis viciifolia) birdsfoot trefoil (Lotus corniculatus), and chicory (Cichorium intybus)

Three temperate forages, sainfoin, birdsfoot trefoil, and chicory, characterized by elevated contents of plant secondary compounds, were compared to a ryegrass-clover mixture (control) in dried (Experiment 1) and ensiled form (Experiment 2) in their palatability and nutritional value. Palatability was measured in adult wethers (n = 6) allowed to choose between the familiar control forage and one of the three test plants. Palatability index was calculated from differences in intake of control and test plants measured after given times. Generally at first contact, palatability of the unfamiliar plants was low. Lag time until palatability index approached or exceeded a value of 100 was 2-5 d, but could not be related to the content of condensed tannins. Sainfoin had a high palatability, the highest content of condensed tannins (77.4 +/- 10.23 g/kg DM), a high content of duodenally utilisable crude protein (94.7 +/- 16.87 g/100 g CP), and a high content of metabolizable energy (9.5 +/- 0.38 MJ ME/kg DM), making this plant most promising for various purposes including anthelmintic action.     

Nitrogen fixation in the rhizosphere and rhizoplane of barley

Summary Aerobic and anaerobic N₂-fixing bacteria developed in the rhizosphere of barley seedlings and exhibited N₂ase activity when seedlings were grown in sterilized sand-nutrient cultures containing low levels of combined nitrogen. The source of the N₂-fixing bacteria appeared to be the seed. Average daily rates up to 0.9 μmoles C₂H₄ h⁻¹ g⁻¹ dry root tissue were measured, but the intensity of the activity was affected by moisture levels and concentration of combined N in the rhizosphere. Removal and washing of the roots did not remove the activity, and roots remained active even after surface-sterilization. An unidentified aerobic N₂-fixing bacterium was isolated from the rhizoplane of active barley roots. Inoculation of barley seedlings with the aerobic N₂-fixing bacterium enhanced N₂ase activity of excised roots 10-fold, with average rates of 0.9, 1.1 and 1.3 μmoles h⁻¹ g⁻¹ dry root assayed under pO₂ of 0.01, 0.02 and 0.04 atm respectively. The aerobic N₂-fixing bacterium also exhibited N₂ase activity when inoculated into the rhizosphere of oat, rice and wheat seedlings. Microscopic observations of sterilized live and stained barley roots suggest that the aerobic N₂-fixing bacterium is an endophyte which infects root tissue and metamorphoses into vesicle-like structures.     

Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules.

Bacteroid differentiation was examined in developing and mature alfalfa nodules elicited by wild-type or Fix- mutant strains of Rhizobium meliloti. Ultrastructural studies of wild-type nodules distinguished five steps in bacteroid differentiation (types 1 to 5), each being restricted to a well-defined histological region of the nodule. Correlative studies between nodule development, bacteroid differentiation, and acetylene reduction showed that nitrogenase activity was always associated with the differentiation of the distal zone III of the nodule. In this region, the invaded cells were filled with heterogeneous type 4 bacteroids, the cytoplasm of which displayed an alternation of areas enriched with ribosomes or with DNA fibrils. Cytological studies of complementary halves of transversally sectioned mature nodules confirmed that type 4 bacteroids were always observed in the half of the nodule expressing nitrogenase activity, while the presence of type 5 bacteroids could never be correlated with acetylene reduction. Bacteria with a transposon Tn5 insertion in pSym fix genes elicited the development of Fix- nodules in which bacteroids could not develop into the last two ultrastructural types. The use of mutant strains deleted of DNA fragments bearing functional reiterated pSym fix genes and complemented with recombinant plasmids, each carrying one of these fragments, strengthened the correlation between the occurrence of type 4 bacteroids and acetylene reduction. A new nomenclature is proposed to distinguish the histological areas in alfalfa nodules which account for and are correlated with the multiple stages of bacteroid development.