Effects of a stay-green mutation on plant nitrogen relations in Lolium perenne during N starvation and after defoliation

The stay-green mutation of the nuclear gene sid results in inhibition of chlorophyll degradation during leaf senescence in grasses, reducing N remobilization from senescing leaves. Effects on growth of Lolium perenne L. were investigated during N starvation (over 18 d) and after severe defoliation, when leaf growth depends on the remobilization of internal N. Rates of dry mater production, partitioning between shoots and roots, and re-partitioning of N from shoots to roots were very similar in stay-green and normal plants under N starvation. Km and Vmax for net uptake of NH4+ were also similar for both genotypes, and Vmax increased with the duration of N deprivation. The mutation had little effect on recovery of leaf growth following severe defoliation, but stay-green plants recommenced NO3- and K+ uptake 1 d later than normal plants. Import of remobilized N into new leaves was generally similar in both lines. However, stay-green plants remobilized less N from stubble compared with normal plants. It was concluded that the sid locus stay-green mutation has no significant adverse effect on the growth of L perenne during N starvation, or recovery from severe defoliation when plants are grown under an optimal regime of NO3- supply both before and after defoliation. The absence of any effect on leaf dry matter production implies that the difference in foliar N availability attributable to this mutation has little bearing on productivity, at least in the short to medium term.     

The relationship between the relative growth rates of the shoot system, number of tillers and mean tiller size in Lolium perenne

The relative growth rate of the shoot system of Lolium perenne may be considered as being made up of two components, the relative growth rate of the increase in the number of tillers and the relative growth rate of the mean tiller. These three relative growth rates were calculated for twenty-two clones of Lolium perenne growing in twenty-eight environments. Analyses of variance showed that differences in the environment were responsible for a greater amount of variation than either differences between clones or the interaction between clonal and environmental influences.
For each clone it was possible to calculate relationships between the relative growth rate of the shoot and its two components as they varied with the environments. The relationships held irrespective of the environmental factor(s) which altered to cause the difference in relative growth rates. In all clones an increase in the relative growth rate of the shoot was found to be due to an increase in the relative growth rates of both components. In seventeen clones the relationship between the increments of the two components was constant. In five of the clones an increase in the relative growth rate of the shoot at low values was due more to an increase in the relative growth rate of the number of tillers than to an increase in the relative growth rate of the mean tiller. At high values the opposite occurred.
These results are discussed in relation to the theory of 'nutritive diversion' and in relation to the proportion of lateral buds which produce tillers.     

Rhizodeposition and C-partitioning of Lolium perenne in axenic culture affected by nitrogen supply and defoliation

This study investigated the effects of N-supply and partial defoliation on C-partitioning, root morphology and soluble rhizodeposition, for Lolium perenne grown in axenic sand culture systems percolated with nutrient solution. Plants were grown for 36 d in nutrient solutions with differing N concentrations (4 mM or 0.02 mM NH₄ ⁺NO₃ ^-), and effects of repeated defoliation to 4 cm were determined. The ‘low N’ supply reduced (P < 0.05) dry matter accumulation, with proportionately increased partitioning to the root systems. Root morphology was also altered at ‘low N’, with development of a finer root system, manifest as increased (P < 0.05) specific root length. Concurrent with these effects on growth of L. perenne, ‘low N’ increased (P < 0.05) exudation of C-compounds from roots on a per g root basis. Defoliation was found to increase exudation (P < 0.05) of soluble compounds for periods of 3-5 d following each cut, at both N-supply rates. The effects of N-supply and defoliation are of importance in understanding the coupling of plant productivity to nutrient cycling in soils with differing N availabilities and for grassland systems which are subject to grazing. This revised version was published online in June 2006 with corrections to the Cover Date.     

High nitrogen supply and carbohydrate content reduce fungal endophyte and alkaloid concentration in Lolium perenne

The relationship between cool-season grasses and fungal endophytes is widely regarded as mutualistic, but there is growing uncertainty about whether changes in resource supply and environment benefit both organisms to a similar extent. Here, we infected two perennial ryegrass (Lolium perenne) cultivars (AberDove, Fennema) that differ in carbohydrate content with three strains of Neotyphodium lolii (AR1, AR37, common strain) that differ intrinsically in alkaloid profile. We grew endophyte-free and infected plants under high and low nitrogen (N) supply and used quantitative PCR (qPCR) to estimate endophyte concentrations in harvested leaf tissues. Endophyte concentration was reduced by 40% under high N supply, and by 50% in the higher sugar cultivar. These two effects were additive (together resulting in 75% reduction). Alkaloid production was also reduced under both increased N supply and high sugar cultivar, and for three of the four alkaloids quantified, concentrations were linearly related to endophyte concentration. The results stress the need for wider quantification of fungal endophytes in the grassland-foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes or increasing N inputs affect the role of endophytes in grassland ecosystems.     

Nitrogen uptake and root morphological responses of defoliated Lolium perenne (L.) to a heterogeneous nitrogen supply

Lolium perenne L. (c.v. Magella) plants were grown under three nutrient treatments for six weeks and then defoliated to test the hypothesis that for their regrowth they could acquire N equally well irrespective of N distribution. Two different N levels were applied; uniform level 1 N (U1), uniform level 2 (U2) and heterogeneous level 2 (H2). A system where the nutrient patch could be applied without barriers to root growth was adopted. A single defoliation to 4 cm height resulted in a reduction in tillering, biomass increment and N uptake at 3 weeks after defoliation. Root growth was reduced by defoliation under all N treatments. Defoliation was found to reduce the proportion of N in the shoots which was derived from root uptake from 7 to 14 days. At 21 days this effect was significant for the plants with a heterogeneously distributed supply only. By the end of the regrowth period, the undefoliated plants from H2 had a shoot biomass and N content equal to that of plants receiving the same total N but supplied homogeneously (U2). However, defoliation reduced the ability of the plant to acquire N from the patch. No preferential root growth was measured into the N-rich patch, but an increased root diameter within the patch was found. Root diameter was reduced by defoliation, coinciding with a reduction in concentration of N in the root tissue. As a result of the increased sink strength of the growing leaves after defoliation, the roots may become a source of carbon and also nitrogen. These responses to an N-rich patch under defoliation could alter a plant's competitive balance in a mixed sward. This revised version was published online in June 2006 with corrections to the Cover Date.     

Responses of plant growth rate to nitrogen supply: a comparison of relative addition and N interruption treatments

This paper investigates the effects of uptake of nitrate and the availability of internal N reserves on growth rate in times of restricted supply, and examines the extent to which the response is mediated by the different pools of N (nitrate N, organic N and total N) in the plant. Hydroponic experiments were carried out with young lettuce plants (Lactuca sativa L.) to compare responses to either an interruption in external N supply or the imposition of different relative N addition rate (RAR) treatments. The resulting relationships between whole plant relative growth rate (RGR) and N concentration varied between linear and curvilinear (or possibly bi-linear) forms depending on the treatment conditions. The relationship was curvilinear when the external N supply was interrupted, but linear when N was supplied by either RAR methods or as a supra-optimal external N supply. These differences resulted from the ability of the plant to use external sources of N more readily than their internal N reserves. These results show that when sub-optimal sources of external N were available, RGR was maintained at a rate which was dependent on the rate of nitrate uptake by the roots. Newly acquired N was channelled directly to the sites of highest demand, where it was assimilated rapidly. As a result, nitrate only tended to accumulate in plant tissues when its supply was essentially adequate. By comparison, plants forced to rely solely on their internal reserves were never able to mobilize and redistribute N between tissues quickly enough to prevent reductions in growth rate as their tissue N reserves declined. Evidence is presented to show that the rate of remobilization of N depends on the size and type of the N pools within the plant, and that changes in their rates of remobilization and/or transfer between pools are the main factors influencing the form of the relationship between RGR and N concentration.     

Effects of genotype and temperature on pollen tube growth in perennial ryegrass (Lolium perenne L.)

Summary Low yield in seed crops of perennial ryegrass is related to low fertilization efficiency and low temperature during anthesis. To study the effect of genotype and temperature on pollen performance, we conducted greenhouse experiments at controlled temperatures. Individual florets of four genotypes that are known to differ in seed production were hand pollinated at four temperatures (14°, 18°, 22°, 26° C) both in vivo and via a semiin-vitro method involving excised florets on agar. Pollen germination and tube growth were determined with UV-fluorescence microscopy and scored in six classes at 2 h after pollination in vitro and after 0.5, 2 and 5 h in vivo. In vitro, both genotype and temperature had a significant effect on the performance of self-pollen. Pollen tube growth increased with temperature. In cross-pollinations, the pistil parent had a significant effect on pollen tube growth, and there was also a significant pistil-by-temperature interaction. In vivo, genotype and temperature significantly affected pollen performance. The genotype-by-temperature interaction was only significant 5 h after pollination. One genotype with low seed yield was pseudoself-compatible and was a relatively poor mother after cross-pollination. The effects of genotype and temperature on the growth of self-pollen might be exploited in a breeding programme.A.G. Stephenson was on a sabbattical leave at SVP in 1987     

Short-term response in leaf metabolism of perennial ryegrass (Lolium perenne) to alterations in nitrogen supply

Nitrogen is a macronutrient present in a wide range of cellular compounds including proteins, nucleic acids, amino acids and lipids. The levels of nitrogen often regulate many aspects of plant metabolism, growth and development. Extensive research has been conducted into the effects of N nutrition in model plants, however relatively little is known about the metabolic response of perennial ryegrass (Lolium perenne) grown under different N-supply conditions. This study aimed to identify key metabolic responses activated rapidly after challenging plants with different levels of N-supply. The metabolic response of the leaves of seven different L. perenne genotypes to three N treatments (low, medium and high levels of N) was characterized using a GC–MS approach. After 24 h it was observed that the levels of amino acids correlated with the levels of N-supply. Furthermore the results indicated that plants experiencing N-limitation accumulated very-long chain fatty acids and precursors of secondary aromatic metabolites while sugar levels were not significantly affected indicating a remobilization of carbon. Plants grown under high levels of N were found to have enhanced levels of inositol and threonic acid which could reflect an alteration of the redox potential under stress. Further analysis of Pearson’s correlation coefficient provided evidence that the chlorophyll metabolism may also be regulated in plants grown at high N concentrations.     

Metabolism and translocation of nitrogen in two Lolium perenne populations with contrasting rates of mature leaf respiration and yield

Barneix, A. J., Cooper, H. D., Stulen, I. and Lambers, H. 1988. Metabolism and translocation of nitrogen in two Lolium perenne populations with contrasting rates of mature leaf respiration and yield. - Physiol. Plant. 72: 631–636.
Several aspects of nitrogen metabolism and transport were investigated to determine whether these processes could account for the observed differences in the dark respiration rate of mature leaves between two populations of Lolium perenne L. cv. S23: GL72 - a slow respiring, high growth rate line, and cv. GL66 - a fast respiring, low growth rate line.
No differences were found in total nitrogen or soluble protein concentrations between the populations, but GL72 showed a higher concentration of soluble amino acids, accounted for mainly by increases in the amounts of asparagine and glutamine. There were no differences in the glutamine synthetase (EC 6.3.1.2) or nitrate reductase (EC 1.6.6.1) activities between populations, but the fast respiring GL66 line showed higher glutamate dehydrogenase (EC 1.4.1.3) and peroxidase (EC 1.11.1.7) activities than GL72. The protein turnover rate, determined from ³H disappearance from leaves labelled with [³H]-acetic anhydride, appeared to be larger in GL66, but the difference was not significant and could not account for the differences in respiration rate.
The apparent extent of ¹⁵N cycling between roots and shoots was low in Lolium compared to other grass species, and there were no differences between the two populations.
It is concluded that the differences in dark respiration rate are not due to differences in demand for ATP by nitrogen assimilatory processes, but may be related to faster leaf senescence in the GL66 population.     

Effects of nutrient supply and competition from other species on root growth of Lolium perenne in soil

Summary In competition experiments with Lolium perenne and Agrostis tenuis on sandy soil with nitrogen supplied and therefore not limiting, it was found that the competitive interactions could be explained in terms of phosphate uptake, and that the ratio of root weight: length was proportional to root density. The effects of competition were then investigated in an experimental system that enabled them to be distinguished from those of nutrient supply. High levels of nutrients specifically stimulated the production of fine laterals whereas competition affected length and weight of the root system of Lolium equally. The ecological implications are discussed in the light of recent physiological work on root responses to nutrient supply. re]19750324     

Polyamine concentrations in four Poa species, differing in their maximum relative growth rate, grown with free access to nitrate and at limiting nitrate supply

Polyamines are thought to play a role in the control of inherent or environmentally-induced growth rates of plants. To test this contention, we grew plants of four grass species, the inherently fast-growing Poa annua L. and Poa trivialis L. and the inherently slow-growing Poa compressa L. and Poa pratensis (L.) Schreb., at three levels of nitrate supply. Firstly, plants were compared when grown with free access to nitrate, allowing the plants to grow at their maximum relative growth rate (RGR_max). Secondly, we compared the plants when grown with relative nitrate addition rates of 100 and 50 mmol N (mol N)^–1 day^–1 (RAR₁₀₀ and RAR₅₀, respectively).The freely-occurring polyamines, spermine, spermidine and putrescine, were separated from their conjugates; the latter were further subdivided into a TCA-soluble and a TCA-insoluble fraction. Each of the three fractions responded differently to the nitrate supply. Under nitrogen limitation, the total concentration of polyamines (free and bound ones together) decreased in both leaves and roots of all Poa species, whereas that in the stem remained more or less the same. These effects were to a large extent determined by the free polyamines. For the conjugates there was more differentiation, both between plant organ and among polyamine structures. A positive correlation between the RGR, LAR (leaf area per plant mass), SLA (leaf area per leaf mass), LMR (leaf mass per plant mass) and SMR (stem mass per plant mass) with the polyamine concentration was found. The RMR (root mass per plant mass) showed a negative one. No significant differences were found between the inherently fast- and slow-growing grass species.The (putrescine)/(spermine + spermidine) ratio in the leaves increased with decreasing nitrate supply, which is associated with a decrease in leaf expansion, accounting for a decrease in LAR and SLA. For the roots, this ratio tended to decrease with decreasing nitrate supply, whereas for the stems the results were somewhat more variable.We found no evidence for a crucial role of polyamines in the determination of inherent variation of growth in spite of a positive correlation of especially the free polyamines with growth parameters.     

Metabolic profiles of Lolium perenne are differentially affected by nitrogen supply, carbohydrate content, and fungal endophyte infection

Lolium perenne cultivars differing in their capacity to accumulate water soluble carbohydrates (WSCs) were infected with three strains of fungal Neotyphodium lolii endophytes or left uninfected. The endophyte strains differed in their alkaloid profiles. Plants were grown at two different levels of nitrogen (N) supply in a controlled environment. Metabolic profiles of blades were analyzed using a variety of analytical methods. A total of 66 response variables were subjected to a principle components analysis and factor rotation. The first three rotated factors (46% of the total variance) were subsequently analyzed by analysis of variance. At high N supply nitrogenous compounds, organic acids and lipids were increased; WSCs, chlorogenic acid (CGA), and fibers were decreased. The high-sugar cultivar 'AberDove' had reduced levels of nitrate, most minor amino acids, sulfur, and fibers compared to the control cultivar 'Fennema', whereas WSCs, CGA, and methionine were increased. In plants infected with endophytes, nitrate, several amino acids, and, magnesium were decreased; WSCs, lipids, some organic acids, and CGA were increased. Regrowth of blades was stimulated at high N, and there was a significant endophyte x cultivar interaction on regrowth. Mannitol, a fungal specific sugar alcohol, was significantly correlated with fungal biomass. Our findings suggest that effects of endophytes on metabolic profiles of L. perenne can be considerable, depending on host plant characteristics and nutrient supply, and we propose that a shift in carbon/N ratios and in secondary metabolite production as seen in our study is likely to have impacts on herbivore responses.     

Nitrogen deficiency inhibits leaf blade growth in Lolium perenne by increasing cell cycle duration and decreasing mitotic and post-mitotic growth rates

Nitrogen deficiency severely inhibits leaf growth. This response was analysed at the cellular level by growing Lolium perenne L. under 7.5 mM (high) or 1 mM (low) nitrate supply, and performing a kinematic analysis to assess the effect of nitrogen status on cell proliferation and cell growth in the leaf blade epidermis. Low nitrogen supply reduced leaf elongation rate (LER) by 43% through a similar decrease in the cell production rate and final cell length. The former was entirely because of a decreased average cell division rate (0.023 versus 0.032 h(-1)) and thus longer cell cycle duration (30 versus 22 h). Nitrogen status did not affect the number of division cycles of the initial cell's progeny (5.7), and accordingly the meristematic cell number (53). Meristematic cell length was unaffected by nitrogen deficiency, implying that the division and mitotic growth rates were equally impaired. The shorter mature cell length arose from a considerably reduced post-mitotic growth rate (0.033 versus 0.049 h(-1)). But, nitrogen stress did not affect the position where elongation stopped, and increased cell elongation duration. In conclusion, nitrogen deficiency limited leaf growth by increasing the cell cycle duration and decreasing mitotic and post-mitotic elongation rates, delaying cell maturation.     

Growth and nitrate uptake properties of plants grown at different relative rates of nitrogen supply. I. Growth of Pisum and Lemna in relation to nitrogen

Abstract The relations between growth and internal nitrogen concentrations were investigated in nonnodulated Pisum sativum L. cv. Marma and Lemna gibba L. grown at relative rates of nitrate-N additions (R_A) varying from 0.03 to 0.27 d ¹(Pisum) and 0.05 to 0.40 d ¹ (Lemna). At R_A≤0.21 d ¹(Pisum) and ≤0.30 d ¹ (Lemna), the relative growth rate (RGR) correlated well with R_A whereas higher R_A was not met by any further increawse in growth rate. The tissue nitrogen concentrations at growth-limiting R_A increased linearly with RGR. The slope of these lines indicate a maximum nitrogen productivity (amount of biomass formed per unit nitrogen and time) of 14.4 g DW g ¹ Nd ¹ for Pisum and 15.9 g DW g ¹ N d ¹ for Lemna. Extrapolation of the plots to RGR=0 yielded intercepts of 10–15 mg N g^?1 DW for Pisum tissue, whereas for Lemna the intercepts were closer to the origin than for Pisum. These intercepts formally define a fraction of the total plant nitrogen that appears not to be active in production of new biomass, her termed ‘non-growth nitrogen’. The partitioning of nitrogen as well as biomass to the roots increased at low R_A, and is discussed in relation to activity of shoots and roots, respectively.     

Nutrition and growth of birch seedlings at varied relative phosphorus addition rates

Birch ( Betula pendula Roth.) was investigated under steady state nutrition and growth at different relative addition rates of phosphorus (R_p). Phosphorus deficiency symptoms appeared on the leaves when the internal phosphorus concentration decreased, but disappeared again under steady state nutrition, independent of the stress level. The increased root/shoot ratio and the exploratory type of root systems developed during the adjustment stage remained under steady state conditions. At nonoptimum and close to optimum relative addition rates, independent of the rate, the phosphorus concentration of the culture solution did not exceed 2 μmol dm⁻³ and was generally < 1 μmol dm⁻³ immediately after phosphorus additions. The phosphorus concentration just before additions was generally < 0.5 μmol dm⁻³. The nutrition/growth relationships were similar to those for nitrogen, with relative growth rate (R_g) closely related to the R_p applied and with a strong linear relationship between internal phosphorus concentration and R_g. Regression was much steeper than that for nitrogen. The slope of the optimum nutrition was attained at a lower phosphorus weight proportion to nitrogen (8–10 P: 100 N) than previously estimated (= 13 P: 100 N), but a higher relative phosphorus requirement was observed under stress conditions. Birch seedlings had a strong tendency to consume phosphorus in excess of immediate requirements with a small effect on growth above optimum. This resulted in rapidly decreasing phosphorus productivity (P_p, growth rate per unit of phosphorus) with increasing internal phosphorus concentrations above optimum.     

Diurnal variation of root respiration rates and nitrate uptake as influenced by nitrogen supply

Root respiration rates of Lolium multiflorum supplied with nitrate or ammonium were measured continuously during several days (Exp. A). Net uptake rate of nitrate was similarly measured by an ion selective nitrate electrode in a system of flowing nutrient solution (Exp. B). Diurnal variation of in vitro nitrate reductase activity and nitrate content of tops and roots were determined (Exp. C). Two levels of irradiance were applied throughout, with day:night of 16:8 h. Root respiration rates showed diurnal patterns, most pronounced in the nitrate treatment, with two peaks appearing about 6 and 16 h after commencement of the photoperiod. Respiration rates were highest in the nitrate treatment and at high irradiance. Respiration rates fell after removal of nitrogen, particularly in the nitrate supplied plant and at high irradiance. Net uptake rate of nitrate exhibited diurnal patterns, often with two peaks occurring at the same times as those of respiration rates. In vitro nitrate reductase activity of tops increased steeply 16 h after commencement of the photoperiod and remained at the high level during the following 8 h of darkness. Nitrate content of tops was highest during the 8 h dark period and fell at the start of the photoperiod. Possible controlling systems of the apparent coincidences of diurnal variation rates, net nitrate uptake and nitrate reduction are discussed.     

Growth and nitrate uptake properties of plants grown at different relative rates of nitrogen supply. II. Activity and affinity of the nitrate uptake system in Pisum and Lemna in relation to nitrogen availability and nitrogen demand

Abstract Net nitrate uptake rates were measured and the kinetics calculated in non-nodulated Pisum sativum L. cv. Marma and Lemna gibba L. adapted to constant relative rates of nitrate-N additions (R_A), ranging from 0.03 to 0.27 d^?1 for Pisum and from 0.05 to 0.40 d^?1 for Lemna, V_max of net nitrate uptake (measured in the range 10 to 100 mmol m^?3 nitrate, i.e. ‘system I’) increased with R_A in the growth limiting range but decreased when R_A exceeded the relative growth rate (RGR), K_m was not significantly related to changes in R_A. On the basis of previous ¹³N-flux experiments, it is concluded that the differences in V_max at growth limiting R_A are attributable to differences in influx rates. Linear relationships between V_max and tissue nitrogen concentrations were obtained in the growth limiting range for both species, and extrapolated intercepts relate well with the previously defined minimal nitrogen concentrations for plant growth (Oscarson, Ingemarsson & Larsson, 1989). Analysis of V_max for net nitrate uptake on intact plant basis in relation to nitrogen demand during stable, nitrogen limited, growth shows an increased overcapacity at lower R_A values in both species, which is largely explained by the increased relative root size at low R_A. A balancing nitrate concentration, defined as the steady state concentration needed to sustain the relative rate of increase in plant nitrogen (R_N), predicted by R_A, was calculated for both species. In the growth limiting range, this value ranges from 3.5 mmol m^?3 (R_A 0.03 d^?1) to 44 mmol m^?3 (R_A 0.21 d^?1) for Pisum and from 0.2 mmol m^?3 (R_A 0.05 d^?1) to 5.4 mmol m^?3 (R_A 0.03 d^?1) for Lemna. It is suggested that this value can be used as a unifying measure of the affinity for nitrate, integrating the performance of the nitrate uptake system with nitrate flux and long term growth and demand for nitrogen.