Linkage map construction in allotetraploid creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is one of the most adapted bentgrass species for use on golf course fairways and putting greens because of its high tolerance to low mowing height. It is a highly outcrossing allotetraploid species (2n=4x=28, A₂ and A₃ subgenomes). The first linkage map in this species is reported herein, and it was constructed based on a population derived from a cross between two heterozygous clones using 169 RAPD, 180 AFLP, and 39 heterologous cereal and 36 homologous bentgrass cDNA RFLP markers. The linkage map consists of 424 mapped loci covering 1,110 cM in 14 linkage groups, of which seven pairs of homoeologous chromosomes were identified based on duplicated loci. The numbering of all seven linkage groups in the bentgrass map was assigned according to common markers mapped on syntenous chromosomes of ryegrass and wheat. The number of markers linked in coupling and repulsion phase was in a 1:1 ratio, indicating disomic inheritance. This supports a strict allotetraploid inheritance in creeping bentgrass, as suggested by previous work based on chromosomal pairing and isozymes. This linkage map will assist in the tagging and eventually in marker-assisted breeding of economically important quantitative traits like disease resistance to dollar spot (Sclerotinia homoeocarpa F.T. Bennett) and brown patch (Rhizoctonia solani Kuhn).     

Effect of wet and dry cycles in calcareous soil on mineral nutrient uptake of two grasses,Agrostis stolonifera L. and Festuca ovina L.

Shoot uptake of mineral nutrients (Ca, Cu, Fe, K, mg, Mn, P, S, Zn) by Agrostis stolonifera L. was compared with Festuca ovina L. under wet and dry cycles. Such conditions are typical for A. stolonifera sites, whereas F. ovina is growing mostly on consistently drier and better-drained soils. Plants were grown in a glasshouse, at controlled temperature and light conditions, using two moisture regimes, one constant at 60% WHC (water holding capacity), one wet/dry fluctuating between 35 and 100% WHC. Above ground and total biomass production was lower under wet/dry treatment than at constant water regime in F. ovina, but did not differ between regimes in A. stolonifera. Shoot uptake of most elements was severely reduced in F. ovina at the wet/dry regime. Shoot uptake and concentrations of most elements studied (Cu, K, Mn, P, S, Zn) were lower (p<0.05) under wet/dry treatment than at constant regime in A. stolonifera and tended to be lower also of Fe and Mg. Differences in biomass production observed are consistent with field evidence that A. stolonifera grows in sites which are periodically flooded but may become quite dry during other periods, and that F. ovina is limited to sites which are consistently drier and better drained. Evidence from the present study, however, does not support any view that alternating wet and dry cycles, as typical of A. stolonifera field sites, would be beneficial to nutrient acquisition of this species but that biomass production may develop normally at the lower uptake of most mineral nutrients measured under the wet/dry regime. Such regimes are decidedly unfavourable to both growth and nutrient acquisition of F. ovina.     

Characterization of 215 simple sequence repeat markers in creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is a versatile, cross-pollinated, temperate and perennial turfgrass species. It occurs naturally in a wide variety of habitats and is also cultivated on golf courses, bowling greens and tennis courts worldwide. Isozymes and amplified fragment length polymorphisms (AFLPs) have been used to determine genetic diversity, and restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPDs) were used to construct a genetic linkage map of this species. In the current report, we developed and characterized 215 unique genomic simple sequence repeat (SSR) markers in creeping bentgrass. The SSRs reported here are the first available markers in creeping bentgrass to date. Eight hundred and eighteen alleles were amplified by 215 SSR loci, an average of 3.72 alleles per locus. Fifty-nine per cent of those alleles segregated in a 1:1 Mendelian fashion (P > 0.05). Twenty-two per cent had a distorted segregation ratio (P ≤ 0.05). These SSR markers will be useful for assessing genetic diversity in creeping bentgrass and will be important for the development of genetic linkage maps and identifying quantitative trait loci. These markers could enhance breeding programmes by improving the efficiency of selection techniques.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated creeping bentgrass (<Emphasis Type="Italic">Agrostis stolonifera</Emphasis> L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration

Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T₁ generation. All independent transformation events carried one to three copies of the transgene, and a majority (60–65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.Abbreviations 2,4-D: 2,4-Dichlorophenoxyacetic acid - bar: Bialaphos resistance gene - GUS: -Glucuronidase - PPT: Phosphinothricin - ubi: Ubiquitin Communicated by J.M. Widholm     

Tolerance ofHolcus lanatus andAgrostis stolonifera to sodium chloride in soil solution and saline spray

Summary Inland and sea cliff populations of bothAgrostis stolonifera L. andHolcus lanatus L. were subjected to soil NaCl treatments, of 100 and 200 mol m⁻³ NaCl, and tolerance examined using plant dry weight data. A parallel experiment subjected them to salt spray treatments of 2.5%, 5% and 10% NaCl in distilled water, and tolerance assessed from leaf damage. Both populations of each species were equally sensitive to soil NaCl. When subjected to salt spray the sea cliff populations however showed marked resistance to leaf damage. Soil salinity resistance and salt spray resistance thus appear to be independent characteristics in these two species.     

Measuring maximum root growth instead of longest root elongation in metal tolerance tests for grasses (Agrostis capillaris,Agrostis delicatula and Agrostis castellana)

Longest root elongation diminished significantly in the three species tested from 6 mm d^-1 to 3 mm d^-1 in 3 weeks. During this period S.D. increased considerably (from 49% to 112%, A. castellana), and accounted on the average for 68% (A. capillaris) till 94% (A. castellana) of the mean. Maximum root growth stabilized at 6 mm d^-1 and showed less variation in the measurements (S.D. 52% of the mean). Growth of the originally longest root approaches zero in all three species, in accordance with the natural cease of growth of roots in grasses fascicular root system. Measuring maximum root growth instead of longest root elongation is proposed for testing metal tolerance of grasses in sequential experiments.     

The effects of nitrogen limitation on the ultrastructure of the cyanobacterium Agmenellum quadruplicatum

The effects of nitrogen limitation on the ultrastructure of the unicellular cyanobacterium, Agmenellum quadruplicatum, were studied by thin sectioning transmission electron microscopy. Nitrogen became limiting for growth 14–15 h after transfer to nitrogen-limiting medium, but cultures retained full viability for at least 45 h. The c-phycocyanin: chlorophyll a ratio and cellular nitrogen content of the culture dropped rapidly after 14–15 h, as a progressive deterioration of major cell structures took place. Phycobilisomes were degraded first, followed by ribosomes and, then, thylakoid membranes. These structures were virtually depleted from the cells within 26 h. Intracellular polysaccharide accumulated in place of the normal cell structures throughout this period. Nitrogen limitation did not affect polyphosphate bodies, carboxysomes, lipid granules, the cell envelope, or the extra-cellular glycocalyx. All of the ultrastructural changes resulting from nitrogen limitation were reversed upon addition of nitrate to a starved culture. Most cell structures were restored within 3 h, and restoration was complete within 9 h.     

Relationship between growth,nitrogen fixation and assimilation in a legume (Medicago sativa L.)

Summary Symbiotic N₂ fixation, NO ₃ ⁻ assimilation and protein accumulation in the shoots were measured simultaneously in alfalfa (Medicago sativa L.) grown in the field or in pots, in order to study how the balance between the two modes of nitrogen nutrition could be influenced by agronomic factors, such as harvest, mineral nitrogen supply and drought stress. During periods of rapid growth, fixation and assimilation may function simultaneously; they are antagonistic at the beginning and at the end of the growth cycle, when the nitrogen requirement of the plant is lower. When nitrogen nutrition does not limit growth, mineral nitrogen supply favours assimilation at the expense of fixation, but does not modify the amount of nitrogen accumulated, which is adjusted to the growth capacity of the plant. After cutting, nitrate assimilation compensated for the decrease in fixation and supplied the plant with the nitrogen required by the regrowth, the proliferation of which determined the fixation recovery. Drought stress decreased N₂ fixation much more than NO ₃ ⁻ assimilation. The latter made growth recovery possible when water supply conditions became normal again. These results suggested the existence of an optimum level of nitrate assimilation, which differed depending on the age of the plants and allowed both maximum growth and fixing activity.     

The effects of salinity on ion concentrations within the root cells of Zea mays L.

Zea mays is a salt-sensitive crop species which in saline (100 mol m^-3 NaCl) conditions suffers considerable growth reduction correlated with elevated Na⁺ and Cl^- concentration within the leaves. To increase understanding of the regulation of ion uptake and transport by the roots in saline conditions, ion concentrations within individual root cortical cells were determined by X-ray microanalysis. There was variation in Na⁺, K⁺ and Cl^- distributions among individual cells, which could not be correlated with their spatial position in the roots. Generally, however, in response to saline growth conditions (100 mol m³ NaCl) Na⁺ and Cl^- were mostly localized in the vacuoles, although their concentrations were also sometimes increased in the cytoplasm and cell walls. The concentration of K⁺ in the cytoplasm was usually maintained at a level (mean 79 mol m^-3) compatible with the biochemical functions ascribed to this ion.Abbreviation (T)AEM (Transmission) analytical electron microscopy     

Effects of Abscisic Acid,Salicylic Acid,Ethylene and Hydrogen Peroxide in Thermotolerance and Recovery for Creeping Bentgrass

Abscisic acid (ABA), salicylic acid (SA), ethylene, and hydrogen peroxide (H₂O₂) may be involved in the regulation of plant responses to heat stress. The objective of this study was to determine whether these signaling molecules are involved in survival at high temperatures in creeping bentgrass (Agrostis stolonifera). We investigated the effects of treatment with ABA, SA, H₂O₂, and ACC (an ethylene precursor) on physiological damage occurring in creeping bentgrass during heat stress (35 °C for 1 month). We also compared the effects of chemical application and the induction of thermotolerance using moderate heat stress (30 °C for 24 h). All of the pre-treatments (heat or chemical) resulted in increased tolerance to prolonged heat stress (1 month) compared to control plants. All treated samples showed more green leaves, decreased membrane leakage and reduced oxidative damage compared to control plants. We then measured changes in the endogenous concentration of these chemical components during heat stress (35 °C) and during recovery after a stress treatment (cooling back to 20 °C). An oxidative burst was detected 5 min after the initiation of heat treatment, with the increase in H₂O₂ being detected primarily in the apoplast of the cells in both leaf and root tissues. Free SA was detected only an hour after the initiation of heat stress, and concentration remained low subsequently. Neither ABA nor ethylene concentrations rose during heat stress, but the concentration of both increased during subsequent cooling. These results suggest that the signaling components of interest are involved in thermotolerance in creeping bentgrass, but that the different chemicals are likely to be involved in separate signaling pathways. An oxidative burst and SA may be bona fide heat stress signals, but ABA and ethylene appear to be involved in signaling pathways in response to recovery from heat stress in this species.     

Controls of nitrogen limitation in tallgrass prairie

Summary The relationship between fire frequency and N limitation to foliage production in tallgrass prairie was studied with a series of fire and N addition experiments. Results indicated that fire history affected the magnitude of the vegetation response to fire and to N additions. Sites not burned for over 15 years averaged only a 9% increase in foliage biomass in response to N enrichment. In contrast, foliage production increased an average of 68% in response to N additions on annually burned sites, while infrequently burned sites, burned in the year of the study, averaged a 45% increase. These findings are consistent with reports indicating that reduced plant growth on unburned prairie is due to shading and lower soil temperatures, while foliage production on frequently burned areas is constrained by N availability. Infrequent burning of unfertilized prairie therefore results in a maximum production response in the year of burning relative to either annually burned or long-term unburned sites.Foliage biomass of tallgrass prairie is dominated by C₄ grasses; however, forb species exhibited stronger production responses to nitrogen additions than did the grasses. After four years of annual N additions, forb biomass exceeded that of grass biomass on unburned plots, and grasses exhibited a negative response to fertilizer, probably due to competition from the forbs. The dominant C₄ grasses may out-compete forbs under frequent fire conditions not only because they are better adapted to direct effects of burning, but because they can grow better under low available N regimes created by frequent fire.     

Interactive effects of salinity and nitrogen on growth and yield of tomato plants

Summary Tomato (Lycopersicon esculentum var. VF 145) plants were grown with Typic Xerofluvents soil in a greenhouse irrigated with recycled nutrient solutions having increasing levels of N and salinity. Positive response of plants to increasing levels of N was obtained at the lowest initial salinity level of 1 dS/m (dS/m=mmho/cm, referenced at 25°C). At the higher initial salinity levels of 5 and 9 dS/m, increasing N was ineffective in counteracting adverse effects on growth and yield caused by the presence of enhanced salt concentrations of the nutrient solution. Total N uptake was linearly correlated with the total water uptake and was severely suppressed by impaired growth associated with the two higher initial salinity levels, irrespective of N levels. The effect of salinity on leaf N concentrations changed over time. Leaf Cl and P concentrations indicated a possible suppressing effect of Cl on P uptake into plant tops.Based on portions of the thesis submitted by the senior author in partial satisfaction of the requirements for the Ph.D. degree in Soil Science. Supported in part by a grant from the Kearney Foundation of Soil Science.     

The control of aldolase in Lemna minor L. in relation to nitrogen deficiency

The loss of activity of aldolase which occurs when Lemna is deprived of nitrogen is shown to be due to the accumulation of a specific inhibitor of aldolase. The inhibitor has been purified 600-fold and has the properties of a low molecular weight protein. The inhibitor is not a proteolytic enzyme and the kinetics of the interaction between aldolase and the inhibitor are reported. The possible physiolgocal significance of the inhibition of aldolase is briefly discussed.     

The interaction of nitrogen assimilation with photosynthesis in nitrogen deficient cells of Chlorella

Nitrogen-limited chemostat cultures of Chlorella fusca var. vacuolata, when given nitrogen in the inorganic forms of nitrate, nitrite and ammonium divert photo-generated electrons, from CO₂ fixation to nitrogen assimilation. Addition of nitrate or nitrite, but not ammonium, stimulates rate of oxygen evolution. All but the most severely nitrogen-deficient culture have increased dark respiration rates after addition of inorganic nitrogen. The nitrite reduction step of nitrogen assimilation is the most light-dependent reaction.Abbreviation DCMU 3-(3, 4-dichloro)-1-1-dimethyl urea - CCCP carbonyl cyanide m-chlorophenylhydrazone     

Regulation of protein synthesis in methylotrophic yeasts: Repression of methanol dissimilating enzymes by nitrogen limitation

The influence of nitrogen limitation on the regulation of the methanol oxidizing enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two methylotrophic yeastsHansenula polymorpha andKloeckera sp. 2201 was studied in continuous culture. When shifted from carbon-limited growth conditions (with a mixture of glucose and methanol as carbon sources) to a nitrogen-limited environment both cultures were found to go through a transition phase where neither enhanced residual concentrations of the nitrogen source nor of one of the two carbon sources could be detected in the supernatant. As soon as nitrogen became a limiting substrate an immediate reorganisation of the cell composition was initiated: protein content of the cells dropped to approximately 40% of its initial value, glycogen was synthesized and the enzyme composition of the cells was changed. The peroxisomal enzymes alcohol oxidase and catalase in both organisms and the two dehydrogenases for formaldehyde and formate in cells ofKloeckera sp. 2201 were subject to degradation (catabolite inactivation). The measured rates of inactivation indicated that in cells ofH. polymorpha this process might be limited to peroxisomes, whereas inKloeckera sp. 2201 the degradation was found to affect peroxisomal as well as cytoplasmic enzymes. In contrast to methanol dissimilating enzymes the net rate of synthesis of hexokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was not affected by this process but those enzymes were synthesized with increased rates.     

Host-genetic control of nitrogen fixation in the legume-Rhizobium symbiosis: complication in the genetic analysis due to maternal effects

Summary In pea cv. Afghanistan a recessive gene sym 6 prevents the full expression of nitrogenase activity in root nodules, induced byRhizobium leguminosarum strain F 13. In contrast, nitrogenase activity is fully expressed in pea cv. Iran. A comparison of the reciprocal hybrids of these two plants showed that the size of the plant was determined by the mother plant (maternal effect). Therefore the shoot weight and the total amount of nitrogen fixed are not suitable as parameters for a genetic analysis. The % nitrogen of the shoot and the specific activity of the nodules per gram of nodules are more reliable, but for practical purposes the specific activity of the nodules expressed per gram of shoot tissue can be used.     

Ectomycorrhizal community composition and function in a spruce forest transitioning between nitrogen and phosphorus limitation

Nitrogen is the main limiting nutrient in boreal ecosystems, but studies in southwest Sweden suggest that certain forests approach phosphorus (P) limitation driven by nitrogen (N) deposition. We added N, P or N + P to a Norway spruce forest in this region, to push the system to N or P limitation. Tree growth and needle nutrient concentrations indicated that the trees are P limited. EMF biomass was reduced only by N + P additions. Soil EMF communities responded more strongly to P than to N. Addition of apatite to ingrowth meshbags altered EMF community composition and enhanced the abundance of Imleria badia in the control and N plots, but not when P was added. The ecological significance of this species is discussed. Effects on tree growth, needle chemistry, and EMF communities indicate a dynamic interaction between EMF fungi and the nutrient status of trees and soils.     

The interaction between Cyprinus carpio L. and Potamogeton pectinatus L. under aquarium conditions

Plants and seedlings of Potamogeton pectinatus were obtained from tubers grown under laboratory conditions. Four plants (mean total length: 14.3 m) and two seedlings (mean height: 10.9 cm) were placed in each of twenty 1001 aquaria illuminated with fluorescent lighting. A 5 cm-thick layer of muddy sediment was then put in each aquarium together with two size-matched fish (mean size classes: 6.8, 14.1 and 23.0 g) of the species Cyprinus carpio. After four weeks, the total length of the plants in the control and small fish aquaria had increased by 71% and 3% respectively, whereas plant total length in the aquaria with medium and large fish had diminished by 33% and 76%, respectively. Few seedlings survived in the presence of the fish. The reduction in plant growth was associated with an increase in water turbidity in all treatments as a result of the benthic feeding habit of C. carpio, and of direct herbivory action in the case of medium- and large-sized fish.